Human sperm incubated in vitro gradually become responsive to inducers of the acrosome reaction. The roles of constituents of the incubation medium are not well understood. These experiments tested the effect of the extracellular pH on sperm acrosomal responsiveness. Sperm were incubated 24 h in media with pH varying between 6.7 and 7.6 and then exposed to progesterone to determine the number of sperm that had become acrosomally responsive. The number of responsive sperm was very low following incubation at pH 6.7-7.0, and increased with the pH over the range 7.0-7.6. Sperm incubated at low pH were not damaged as assessed by motility or viability, and if they were transferred to medium of high pH for 8 h, the inhibition of acrosomal responsiveness was reversed. Inhibition of acrosomal responsiveness at low pH was not due to impaired loss of sperm cholesterol, but was correlated with a reduced intracellular pH. The inhibition of acrosomal responsiveness by media of low pH may result from preventing the normal capacitation-related rise in intracellular pH.