A rapid and gentle procedure has been developed for the separation of spermatozoa from other seminal cells and for the preparation of purified spermatozoa with uniform progressive velocity. The method consists of the filtration of semen through an isotonic. Percoll layer and the subsequent centrifugation of the cell pellet on a preformed continuous Percoll gradient; after fractionation of the density gradient, immotile spermatozoa are recovered at density 1.06 to 1.09, whereas an increase in progressive velocity is measured for spermatozoa collected at higher densities. The usefulness of this simple method as a diagnostic aid in men with infertility and urogenital disease was illustrated by the analysis of two abnormal semen samples. This method for the preparation of homogeneous sperm cell populations might facilitate further (sub)cellular studies under normal and pathologic conditions.