Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) in ovarian follicle cells of the starfish Asterina pectinifera was studied. Protein kinase activity in follicle cell homogenate was activated by cAMP in a dose-dependent manner, and Ka was obtained with 10(-7) M cAMP. The PKA activity required Mg2+ at concentrations between 2 and 10 mM. On Sephacryl S-300 column chromatography of partially purified PKA, Mr of the holoenzyme was estimated to be about 180,000. [2,3-3H]cAMP binding activity also suggested a regulatory subunit of Mr about 50,000. DE-52 column chromatography of the cell extract resolved the enzyme activity into two peaks, which eluted between 0.05 and 0.1 M NaCl (type I), and between 0.15 and 0.25 M NaCl (type II). The type I enzyme was the predominant form of PKA in starfish follicle cells. In a cell-free system, a 70 kDa protein was phosphorylated during incubation with [gamma-32P]ATP in the presence of cAMP. These results suggest that PKA stimulates the phosphorylation of a 70 kDa protein following an increase in the level of cAMP.