Heparin facilities the acrosome reaction in bull and rabbit sperm. It binds to sperm saturably, and the binding affinity and the susceptibility to heparin are related to fertility. Isolation of heparin-binding proteins from washed ejaculated sperm reveals proteins mostly in the 14-18 kilodalton (kD) range. Seminal plasma inhibits heparin-induced capacitation, possibly by binding and inactivating heparin. These experiments focused on isolating and characterizing heparin-binding proteins in seminal plasma. Semen samples from 6 bulls were pooled, centrifuged twice for 10 min at 500 xg and finally at 2000 xg for 10 min. The supernatant was ultrafiltered on an Amicon YM-5 (5 kD cutoff) membrane against 40 mM Tris with 2 mM Ca2+ plus protease inhibitors. Five mg of protein were applied to a 1.5 x 10 cm column of heparin-Sepharose. Bound proteins were eluted with 200 ml of a 0 to 2 M NaCl gradient at 0.33 ml/min collecting 3 ml fractions. Two major peaks eluted at 0.12 and 0.46 M NaCl. Those were subjected to SDS-PAGE under reducing conditions. The 0.12 M peak was composed of small (mol wt 15.5 and 16.5 kD) polypeptides which were similar to those found on sperm. The 0.46 M peak included those polypeptides plus a 19.5 kD polypeptide and its trailing edge had a 29.5 kD polypeptide. These polypeptides may regulate capacitation by viture of their ability to bind heparin.