The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.