Summary.In order to ensure fertility, mammalian spermatozoa have to undergo acrosome reaction, the most obvious morphological change during this being the exposure of the inner acrosomal membrane. In the present study, the acrosome‐reacted human spermatozoa were successfully separated without loss of viability by using cell affinity chromatography on Concanavalin A (Con A) Sepharose. Con A demonstrated affinity for both the intact and the acrosome‐reacted spermatozoa regardless of their viability; the latter, however, gave higher affinity than the former against Con A. Prior to the column chromatography, the immotile spermatozoa and the seminal plasma were excluded by means of a modified swim‐down procedure and the resulting spermatozoa were subsequently immobilized by slow rate cooling in ice‐cold water. Cell affinity chromatography was performed at 4 °C. To prevent mechanical trapping of the spermatozoa among the packed gel beads, the column was interconnected with a reservoir, the vertical drive of which was allowed to lose the gel bed and thereby release the trapped spermatozoa. Stepwise competitive elution with 5.0 μM mannose and 25% heat‐inactivated human serum was capable of separating the intact spermatozoa and the acrosome‐reacted spermatozoa from each other. The acrosome reaction rate of sperm fraction which was adsorbed to Con A Sepharose and eluted with 25% serum was found to be 83±2.3%, and motility and viability of these fractions were measured to be 80±6.3% and 83±7.6%, respectively (n= 8, mean±SD). The status of the acrosome in a final preparation (motility 92%, acrosome reaction rate 88%) was observed by scanning electron microscopy, and 81% spermatozoa lost their acrosome cap.Acrosome‐