AbstractMammalian spermatozoa have been shown to possess cAMP‐dependent protein kinase (A‐PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A‐PK, stimulated over a concentration range of 10−9to 10−5M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10−6M), testicular spermatozoa had significantly greater amounts of cAMP‐dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP‐40‐soluble protein extracts revealed the presence of two substrate proteins (Mr= 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (Mr= 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP‐modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during ep