Previous studies have established that the mammalian sperm acrosome reaction (AR) is dependent upon an influx of extracellular Ca2+, but the involvement of a mobilizable store of intracellular Ca2+ has not been shown. In many other cells, the endoplasmic reticulum is the site of such a Ca(2+)-store. Here, we show that thapsigargin, a highly specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase Ca(2+)-pump (and thus a mobilizer of intracellular Ca2+) in other cells, can initiate the AR in capacitated human sperm. Thapsigargin at concentrations from 50-500 nM significantly increased the AR to the same extent when incubated with capacitated sperm for 1 min (assayed by indirect immunofluorescence). Transmission electron microscopy confirmed the occurrence of normal morphology in the AR initiated by thapsigargin. Thapsigargin (200 nM) did not initiate the AR in noncapacitated sperm. Initiation of the AR by thapsigargin apparently requires an influx of Ca2+ since 1 min preincubation with the calcium channel blockers La3+ (250 microM) or Ni2+ (250 microM) prior to addition of thapsigargin completely inhibits AR-initiation. Mobilization of an intracellular Ca(2+)-store by thapsigargin in capacitated human sperm may lead to an influx of extracellular Ca2+ and subsequently the AR. Putative sites for thapsigargin-sensitive intracellular Ca(2+)-stores in human sperm include the cytoplasmic droplet, the sperm nucleus and the acrosome.