The biochemical characteristics of cyclic AMP-dependent protein kinase in calf-snout epidermis were investigated. The activity of cyclic AMP-dependent protein kinase was higher in the lower layer than the upper layer of epidermis. The supernatant of homogenates of the lower layer of calf-snout epidermis was fractionated by DEAE-cellulose chromatography and contained two major peaks of protein kinase activity stimulated by cyclic AMP. This chromatographic pattern is similar to that referred to as Type I and Type II of cyclic AMP-dependent protein kinase in bovine muscle. Both peaks of cyclic AMP-dependent protein kinase in calf-snout epidermis could phosphorylate keratin polypeptides in vitro. The phosphorylation reaction was activated by cyclic AMP and inhibited by a heat-stable inhibitor of cyclic AMP-dependent protein kinase. When Type II enzyme of cyclic AMP-dependent protein kinase was incubated with [gamma-32P]ATP in the absence of substrates, such as histone or keratin polypeptides, the 54,000 dalton protein was phosphorylated and this autophosphorylation was inhibited by the addition of 10 microM cyclic AMP. These results suggest that cyclic AMP-dependent protein kinase in calf-snout epidermis has properties similar to those in bovine muscle and plays an important role in the phosphorylation of keratin polypeptides in calf-snout epidermis.