The basis for the unusual properties of the catalytic subunit (C) of ram sperm cAMP-dependent protein kinase was investigated. Ram sperm C was purified and found by mass spectrometry (MS) to be approximately 890 Da smaller than Calpha, the predominant somatic isoform. Partial internal amino acid sequence from ram sperm C was an exact match to that of bovine Calpha, but differed from the predicted sequences for the Cbeta and Cgamma isoforms. MS analysis of 2-nitro-5-thiocyanatobenzoic acid fragments showed that the mass difference originated in the amino-terminal region. A unique blocked amino-terminal fragment was isolated from sperm C and sequenced by a combination of tandem mass spectrometry and Edman degradation of a subfragment. The results revealed that the amino-terminal myristate and the first 14 amino acids of Calpha are replaced by an amino-terminal acetate and six different amino acids in sperm C. The predicted mass difference due to these changes is 899 Da. The region of homology between sperm C and Calpha begins at the exon 1/exon 2 boundary in Calpha, suggesting that sperm C results from use of an alternate exon 1 in the Calpha gene. The different amino terminus of sperm C may be related to a unique requirement for localization of the "free" C subunit within the sperm flagellum.