High-quality motile human spermatozoa were obtained following treatment of semen by simultaneous swim-up into medium and swim-down into an isotonic 40% Percoll solution.This procedure was significantly better than the swim-up method and comparable to discontinuous Percoll gradient centrifugation. Recovery rates of motile sperm were 35% for swim-up, 65% for Percoll gradient centrifugation, and 73% for swim-up/swim-down.The swim-down sperm was inferior to the swim-up sperm in its upward migration capacity but superior in morphology. Spermatozoa obtained by the swim-up/swim-down procedure demonstrated fertilizing ability in IVF, and clinical pregnancies were established. The simultaneous swim-up/swim-down procedure offers an alternative efficient method of simple separation of high-quality motile sperm for various assisted reproduction techniques.