Plasma membrane proteins were extracted either from epididymal sperm after incubation with ampullary gland secretion or from uterine sperm derived from surgically treated males belonging to the following groups: TX, excision of all accessory sex glands (ASG); AGX, bilateral excision of ampullary glands; AG, excision of all ASG except ampullary glands; and SH, sham-operated. Total membrane protein, glycoprotein, and SDS-PAGE of individual polypeptide subunits were quantified. After incubation with ampullary gland secretion, both protein and glycoprotein concentrations of epididymal sperm membrane were increased. The protein profile was also significantly altered, with the removal of the 43- and 71-kD subunits and the addition of the 36- and 50-kD subunits. The in vitro results confirmed this proteolytic effect of ampullary gland and other ASG on the 43- and 71-kD subunits, despite a reduction in membrane protein concentration. Modification of the 17-, 20-, 25-, 28-, 56-, and 66-kD proteins were also observed. This report is the first demonstration that the ampullary gland is capable of modifying proteins on the sperm surface.