Prophospholipase A₂ (proPA₂) has been isolated from human spermatozoa after acid extraction and chromatography on hydrophobic WP-Butyl (C₄) and ion-exchange (SP 5PW) columns. The addition of benzamidine, a noncompetitive synthetic trypsin inhibitor, to semen samples has kept a portion of the sperm phospholipase A₂ (PA₂) in its zymogen form and allowed its isolation after acid extraction. When radioactive phosphatidylcholine (PC) or phosphatidylethanolamine (PE) were used as substrates, an identical elution profile of this enzyme was obtained on a C₄ column. The proenzyme was separated from active PA₂ on the C₄ column. Human sperm proPA₂ exhibited a less cationic charge than active PA₂ on the SP 5PW column. Porcine pancreatic proPA₂ had the same chromatographic behavior on high performance liquid chromatography (HPLC) (SP 5PW) as human sperm proPA₂. The purification procedure resulted in the isolation of proPA₂ which, upon activation by proteolysis, presented the same chromatographic elution profile on HPLC as active PA₂ of human spermatozoa and porcine pancreas. Thus, a zymogen form of PA₂ exists in human spermatozoa.