A newly developed method for the isolation of human motile spermatozoa using density-gradient centrifugation was compared with the traditionally used Percoll technique.Sperm samples were divided into two equal aliquots, which were purified with either the traditionally performed Percoll technique or a new alternative based on polysucrose/Optiprep media. For each sample the isolation was performed during the same run of the centrifuge.The average recovery of progressively motile spermatozoa with the polysucrose/Optiprep method was significantly higher (48 +/- 7%) than with the Percoll method (38 +/- 6%) (n = 18). The average percentage of motile spermatozoa and the motility score were similar in the purified preparations.The new polysucrose/Optiprep-based density-gradient centrifugation technique for the isolation of motile human spermatozoa is as good as the traditionally used Percoll method and may replace it in connection with assisted reproduction techniques.