The catalytic subunits of protein kinase A are transcribed in all mouse tissues from two distinct genes that code for the Calpha and Cbeta isoforms. Alternative promoters exist for the Cbeta gene that are used in a tissue-specific fashion and give rise to variants that differ in their amino-terminal sequences. We have characterized an alternative promoter that is present in the first intron of the Calpha gene and is transcriptionally active in male germ cells. Transcription from this promoter is coincident with the appearance of pachytene spermatocytes and leads to a Calpha protein (Calpha2) that contains a distinctive 7 amino acid amino-terminus differing from the 14 amino acid amino-terminus of Calpha1. The Calpha2 protein does not contain the myristylation signal present on Calpha1 and migrates at a lower molecular weight on SDS/PAGE gels. By Western blotting, we estimate that most or all of the Calpha protein present in mature sperm is Calpha2. The amino-terminal sequence of Calpha2 is similar to that of ovine sperm C as previously reported [San Agustin, J. T., Leszyk, J. D., Nuwaysir, L. M. & Witman, G. B. (1998) J. Biol. Chem. 273, 24874-24883], and we show by cDNA cloning that human sperm also express a highly related Calpha2 homolog. The Calpha2 subunit forms holoenzymes with either RIIalpha or RIalpha, and both activate at the same concentration of cyclic nucleotide. Because protein kinase A is thought to play a pivotal role in sperm motility and capacitation, the distinctive biochemical properties of the unmyristylated Calpha2 may be essential for fertility in the male.