The authors compared a simple, two-layer Percoll density gradient technique with the swim-up technique for semen preparation in 128 men. In samples from normospermic (n = 55), oligospermic (n = 26), and asthenospermic (n = 29) men, the Percoll technique significantly improved yield, percent motility, and absolute number of motile sperm recovered, but in samples from oligoasthenospermic men (n = 18), only percent motility was improved. The Percoll density gradient also selected sperm with markedly improved function as assessed by both the sperm penetration assay and the fertility index. In 37 samples negative on the sperm penetration assay when processed with the swim-up technique, 19 (51%) became positive when processed with the Percoll technique. The Percoll density gradient is an improved method for semen manipulation as it allows greater recovery of sperm with higher motility and improved sperm function.