We have compared the rat protamine 2 gene sequence (rP2) to that of the mouse protamine 2 (mP2) gene. The sequence encompasses 435 nucleotides of the coding region which includes an intron of 120 nucleotides, 461 nucleotides 5' to the coding sequence and 181 bases 3' to it. In the mouse the protamine 2 gene is abundantly transcribed and translated. The mP2 protein is initially synthesized as a precursor and then proteolytically processed to yield the mature protein. In contrast, in the rat, protamine 2 transcripts are present at 2-5% that found in the mouse and the mature protein has never been detected in spermatozoa. Although there is 92% nucleotide similarity between rat and mouse genes and 91% similarity of the predicted amino acid sequences, in vitro runoff transcription assays performed in either rat or mouse testis-derived transcription systems reveal that the rP2 promoter is only 30% as efficient a promoter as the mP2 promoter. Analyses of total sperm basic nuclear proteins extracted from epididymal sperm using a monoclonal antibody specific for protamine 2 suggest that the rat P2 mRNA is translated in vivo but is not properly processed. These results suggest that the lowered transcription rate and altered processing sites of the rat protamine 2 gene are likely to contribute to the lack of protamine 2 in rat spermatozoa.