It was shown previously by us that cAMP-dependent protein kinase activity in the cellular slime mold Dictyostelium discoideum increased during the early stages of development. Results from other laboratories showed that during the subsequent stage of cell differentiation and positioning, the accumulation of a number of prespore mRNAs and proteins (but not prestalk mRNAs and proteins) was dependent upon cAMP. The present communication describes the cellular distribution of the cAMP-dependent protein kinase at that stage of development. Pseudoplasmodia were disrupted, and prespore cells were separated from prestalk cells by sedimentation through a Percoll gradient. Prespore cells had approximately 4-5 times as much of both the catalytic and regulatory subunits of the cAMP-dependent protein kinase as did the prestalk cells. That the increase of cAMP-dependent protein kinase during development reflected de novo synthesis of the enzyme in both prespore and prestalk cells was demonstrated on the basis of [(3)H]leucine incorporation into the regulatory subunit. The findings are consistent with a role of the cAMP-dependent protein kinase in mediating the effects of cAMP on the synthesis of prespore-specific mRNAs and proteins at the stage at which cAMP appears to be required for the cell type-specific syntheses.