The pH of the hamster sperm acrosome was estimated by a method based on the distribution of monoamines between membrane enclosed volumes maintaining pH gradients. A fluorescent amine, 9-aminoacridine, was used to permit both microscopic and fluorometric measurements of amine distribution. Cauda epididymal hamster sperm incubated with 9-aminoacridine accumulated the amine in the acrosomal volume. In the presence of NH4Cl or the ionophore Nigericin (compounds which discharge pH gradients) 9-aminoacridine fluorescence disappeared from the acrosome. Amine distribution between the acrosome and external volume was estimated by fluorometric measurement of sperm filtrates in the presence and absence of NH4Cl and Nigericin. These values, together with an estimated acrosomal volume of 0.4mu3 were used to calculate an acrosomal pH of less than 5. In addition, an acrosomal pH of 5 or less was obtained with 14C-methylamine. We suggest that such an acidic acrosomal pH of 5 or less could serve to inhibit the activation or autoactivation of the acrosomal zymogen proacrosin to acrosin, a trypsin-like enzyme involved in fertilization.