Bovine seminal platelet-activating factor acetylhydrolase was isolated to >90% purity in a single step using a butyl sepharose column. The procedure involves the elution of the activity by use of an ethanol gradient. Protein binds readily to the resin in the absence of high ionic strength and elutes as a peak centered at 30% ethanol. Approximately 2 mg (by Bradford; 5 mg by weight) of the enzyme can thus be easily obtained from 9 ml of seminal plasma. The specific activity of the purified protein was 22 micromol/minute/mg. About 10% of the loaded activity systematically passed unadsorbed through the column, even after repassing. Most of this activity, however, was attributed to the same or a very similar enzyme that cross-reacts with polyclonal antibodies directed against the highly purified platelet-activating factor acetylhydrolase. The enzyme was acid-labile but very resistant to freezing and lyophilization. This purification procedure should constitute a valuable asset to investigators interested in platelet-activating factor and platelet-activating factor acetylhydrolase roles in reproductive biology.