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| 1st(1st leg) | Recreativo Huelva | - | Sporting Gijón | 1:1 (1:0) | ||
| 2nd (2nd leg) | Sporting Gijón | - | Recreativo Huelva | 2:4 (1:1, 1:1, 1:1) pso | ||
| 1st(1st leg): 27/10/2009 19:00 | 2nd (2nd leg): 10/11/2009 19:00 | Winner: Recreativo Huelva | ||||
| 1st(1st leg) | CF Atlético Ciudad | - | Sevilla FC | 2:4 (2:4) | ||
| 2nd (2nd leg) | Sevilla FC | - | CF Atlético Ciudad | 5:1 (0:0) | ||
| 1st(1st leg): 27/10/2009 19:00 | 2nd (2nd leg): 10/11/2009 19:00 | Winner: Sevilla FC | ||||
| 1st(1st leg) | UD Marbella | - | Atlético Madrid | 0:2 (0:1) | ||
| 2nd (2nd leg) | Atlético Madrid | - | UD Marbella | 6:0 (4:0) | ||
| 1st(1st leg): 27/10/2009 19:00 | 2nd (2nd leg): 10/11/2009 18:00 | Winner: Atlético Madrid | ||||
| 1st(1st leg) | AD Alcorcón | - | Real Madrid | 4:0 (3:0) | ||
| 2nd (2nd leg) | Real Madrid | - | AD Alcorcón | 1:0 (0:0) | ||
| 1st(1st leg): 27/10/2009 21:00 | 2nd (2nd leg): 10/11/2009 19:00 | Winner: AD Alcorcón | ||||
| 1st(1st leg) | Real Zaragoza | - | Málaga CF | 1:1 (1:0) | ||
| 2nd (2nd leg) | Málaga CF | - | Real Zaragoza | 0:0 (0:0) | ||
| 1st(1st leg): 28/10/2009 19:00 | 2nd (2nd leg): 10/11/2009 19:00 | Winner: Málaga CF | ||||
| 1st(1st leg) | Xerez CD | - | CA Osasuna | 1:2 (0:2) | ||
| 2nd (2nd leg) | CA Osasuna | - | Xerez CD | 1:0 (0:0) | ||
| 1st(1st leg): 28/10/2009 19:00 | 2nd (2nd leg): 10/11/2009 19:00 | Winner: CA Osasuna | ||||
| 1st(1st leg) | Rayo Vallecano | - | Athletic Bilbao | 2:0 (2:0) | ||
| 2nd (2nd leg) | Athletic Bilbao | - | Rayo Vallecano | 2:2 (1:0) | ||
| 1st(1st leg): 28/10/2009 19:00 | 2nd (2nd leg): 11/11/2009 20:00 | Winner: Rayo Vallecano | ||||
| 1st(1st leg) | RC Celta | - | CD Tenerife | 2:1 (2:1) | ||
| 2nd (2nd leg) | CD Tenerife | - | RC Celta | 0:1 (0:0) | ||
| 1st(1st leg): 28/10/2009 19:00 | 2nd (2nd leg): 10/11/2009 19:00 | Winner: RC Celta | ||||
| 1st(1st leg) | Real Murcia | - | Deportivo La Coruña | 0:1 (0:0) | ||
| 2nd (2nd leg) | Deportivo La Coruña | - | Real Murcia | 0:0 (0:0) | ||
| 1st(1st leg): 28/10/2009 19:00 | 2nd (2nd leg): 10/11/2009 19:00 | Winner: Deportivo La Coruña | ||||
| 1st(1st leg) | Hércules CF | - | UD Almería | 2:1 (0:1) | ||
| 2nd (2nd leg) | UD Almería | - | Hércules CF | 0:1 (0:0) | ||
| 1st(1st leg): 28/10/2009 19:00 | 2nd (2nd leg): 12/11/2009 20:00 | Winner: Hércules CF | ||||
| 1st(1st leg) | Getafe CF | - | Espanyol Barcelona | 2:0 (0:0) | ||
| 2nd (2nd leg) | Espanyol Barcelona | - | Getafe CF | 1:1 (1:1) | ||
| 1st(1st leg): 28/10/2009 19:00 | 2nd (2nd leg): 11/11/2009 19:00 | Winner: Getafe CF | ||||
| 1st(1st leg) | CD Alcoyano | - | Valencia CF | 0:1 (0:1) | ||
| 2nd (2nd leg) | Valencia CF | - | CD Alcoyano | 2:2 (2:1) | ||
| 1st(1st leg): 28/10/2009 19:00 | 2nd (2nd leg): 10/11/2009 18:00 | Winner: Valencia CF | ||||
| 1st(1st leg) | CD Leonesa | - | FC Barcelona | 0:2 (0:1) | ||
| 2nd (2nd leg) | FC Barcelona | - | CD Leonesa | 5:0 (0:0) | ||
| 1st(1st leg): 28/10/2009 21:00 | 2nd (2nd leg): 10/11/2009 21:00 | Winner: FC Barcelona | ||||
| 1st(1st leg) | Real Valladolid | - | RCD Mallorca | 2:1 (1:1) | ||
| 2nd (2nd leg) | RCD Mallorca | - | Real Valladolid | 1:0 (1:0) | ||
| 1st(1st leg): 29/10/2009 20:00 | 2nd (2nd leg): 11/11/2009 20:00 | Winner: RCD Mallorca | ||||
| 1st(1st leg) | UD Salamanca | - | Racing Santander | 1:0 (0:0) | ||
| 2nd (2nd leg) | Racing Santander | - | UD Salamanca | 4:1 (2:1) | ||
| 1st(1st leg): 29/10/2009 20:00 | 2nd (2nd leg): 11/11/2009 20:00 | Winner: Racing Santander | ||||
| 1st(1st leg) | Unión Deportiva Puertollano | - | Villarreal CF | 1:1 (1:0) | ||
| 2nd (2nd leg) | Villarreal CF | - | Unión Deportiva Puertollano | 1:0 (0:0) | ||
| 1st(1st leg): 29/10/2009 20:00 | 2nd (2nd leg): 10/11/2009 18:00 | Winner: Villarreal CF | ||||
