The distribution of calcium was determined in the vas deferens of the guinea pig using 45Ca electron microscopic autoradiography of rapidly frozen, freeze-dried, and embedded tissue. A selective accumulation of calcium at the plasma membrane and SR was observed in vas deferens that had been incubated in 45Ca for 65–85 min prior to rapid freezing. Rinsing the tissue in nonradioactive calcium for 6 min prior to rapid freezing significantly altered the distribution of calcium among the plasma membrane, mitochondria, and cytoplasmic matrix. The influence of species on the observed distribution of calcium was also examined. The distribution of calcium in the guinea pig was deferens was not significantly different from that in the rabbit vas deferens when the tissues were prepared under identical conditions.