The effect of heparin-induced capacitation on the intracellular pH (pHi) of individual bovine sperm was determined with image analysis. Sperm were loaded with the acetoxymethyl ester of the pH sensitive fluorescent indicator, 2',7'-bis(carboxyethyl)-5(6)-carboxy-fluorescein (BCECF). The pHi of 5303 sperm was evaluated from a total of five bulls at .5, 2, 3, 4, and 5 h of incubation. The pHi did not differ between the sperm head and mid-piece (P > 0.05). An increase in sperm head pHi was seen in heparin-treated sperm at 3, 4, and 5 h of incubation relative to sperm incubated without heparin (control, P < 0.05). At 5 h of incubation, the pHi in heparin-treated sperm was 6.92 +/- 0.07, while control-treated sperm pHi was 6.70 +/- 0.03. Initially a normal frequency distribution was seen for sperm pHi in both heparin- and control-treated sperm. As the incubation progressed, the frequency distribution began to skew towards higher pHi in both samples but was more dispersed for the heparin-treated sperm. Following an NH4Cl-induced alkaline load, the pHi of both control- and heparin-treated sperm recovered toward the resting pHi with a half-time of recovery of 1.5-1.7 min. The recovery of sperm pHi was not due to leakage of NH4+ into sperm because recovery also occurred with trimethylamine. The instantaneous velocity of the pHi recovery (v(i)) was dependent on pHi and decreased as pHi decreased. Capacitation by heparin was associated with an 81% decrease in v(i) at a pHi of 7.00, but there was no effect of capacitation on the proton buffering power of the sperm, which was 87 +/- 8 mM/pH unit. Results demonstrate that both the regulation of pHi and resting pHi were altered during capacitation of bovine sperm by heparin.