The level of the regulatory subunit of the cAMP-dependent protein kinase from Dictyostelium discoideum was analyzed in subcellular fractions of cells at various stages of development by Western blotting. The protein was found only in the cytosolic fraction. A small amount of regulatory subunit was present in vegetative cells, and its level increased sharply during the first hours of aggregation; a further increase also occurred during culmination. Analysis of mature spores and of the stalky mutant HL 65 revealed that the protein is present only in prespore cells.