The soluble cAMP-dependent protein kinase activities of two estrogen receptor-containing (MCF-7 and ZR-75-1) and two estrogen receptor-lacking (BT-20 and MDA-MB-231) established human mammary tumor cell lines were analyzed by DEAE-cellulose chromatography and photoaffinity labeling with 8-azido-[32P]cAMP. Predominantly, type I isoenzyme was present in MDA-MB-231 cells, type II protein kinase was the main form in ZR-75-1 and BT-20 cells; whereas MCF-7 cytosols contained equal amounts of both protein kinase types. No correlations between estrogen receptor content and cAMP-dependent protein kinase holoenzyme ratios of isoenzymes were found. A distinctly greater heterogeneity of charge isomers of cAMP-binding proteins (regulatory subunits) was observed in estrogen receptor-containing cells.