Endogenous phosphorylation was measured in soluble fractions at four stages of Dictyostelium discoideum development. A peak of activity occurred at the slug stage, coincident with the appearance of cyclic AMP-dependent protein kinase. After partial purification by DE-52 cellulose and Sephacryl S-300 chromatography, cyclic AMP dependency of six cytosolic proteins was observed, with apparent subunit molecular weights of > 200,000, 110,000, 107,000, 91,000, 75,000, and 69,000. Phosphorylated bands at 107,000 and 91,000 MW were found to be specific for a partially-purified catalytic subunit prepared from the Dictyostelium holoenzyme form.