The pattern of labelled proteins synthesized and secreted in vitro by the adult mouse epididymis was studied by one- and two-dimensional polyacrylamide gel electrophoresis. The presence of a major 24 kDa protein synthesized and secreted in a tissue-specific manner by the caput epididymidis was detected. For this molecular weight, two-dimensional analysis indicated several proteins including two polypeptides (pI 8.4 and 8.8) whose expression is under androgenic control. Partial amino acid sequence analysis showed a complete N-terminal identity between these two peptides. A polyclonal monospecific immune serum was raised against the two proteins. Only purified immunoglobulins precipitated them, showing that immunological affinity is restricted to these two proteins in the epididymis. Indirect immunofluorescence assay revealed specific binding of antibodies on the acrosomal region of spermatozoa isolated from the caput, corpus or cauda epididymides. Testicular spermatozoa were not labelled under the same conditions. To investigate the physiological role of androgens in the synthesis and secretion of the 24 kDa proteins, tissue slices of epididymides from adult mice which had been castrated, or castrated then injected with testosterone were incubated with [35S]methionine. Castration and testosterone replacement kinetics revealed that alterations in 24 kDa protein synthesis follow immediately upon androgenic privation and replacement.