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Date: 19 September 2000
Summary
Semi-final 1
| Rank | Name | Nation | Time |
| 1 | Michael Klim | AUS | 48.80 |
| 2 | Alexander Popov | RUS | 48.84 |
| 3 | Denis Pimankov | RUS | 49.43 |
| 4 | Jose Martin Meolans | ARG | 49.66 |
| 5 | Duje Draganja | CRO | 49.67 |
| 5 | Lorenzo Vismara | ITA | 49.67 |
| 7 | Salim Iles | ALG | 49.70 |
| 8 | Christian Troeger | GER | 49.80 |
| Rank | Name | Nation | Time |
| 1 | Pieter van den Hoogenband | NED | 47.84 |
| 2 | Lars Froelander | SWE | 48.93 |
| 3 | Neil Walker | USA | 49.04 |
| 4 | Gary Hall Jr. | USA | 49.13 |
| 5 | Chris Fydler | AUS | 49.55 |
| 6 | Attila Zubor | HUN | 49.58 |
| 7 | Roland Schoeman | RSA | 49.84 |
| 8 | Gustavo Borges | BRA | 49.93 |