A 500-base cDNA encoding the bovine protamine was used for hybridization experiments with total RNA prepared from testes of prepubertal and sexually mature bulls and from pachytene spermatocytes and spermatids isolated from mature testes. The mRNA for protamine was first detected in the 7-month old testis containing 10-15% of round spermatids but was absent in testes of younger animals containing only diploid spermatogenic cells. Hybridization of the protamine cDNA with the RNA of isolated spermatids of the mature testis resulted in a prominent hybridization signal, while the faint signal obtained with the RNA of pachytene spermatocytes was found to be due to contamination of the cell preparation by spermatids. As demonstrated by in situ hybridization on testis-sections the transcripts are confined to the central cell layers of the tubuli seminiferi corresponding to the spatial arrangement of postmeiotic spermatogenic cells. The results indicate that the protamine gene in the bull is postmeiotically expressed and the mRNA is synthesized as a 680 nucleotide long molecule.