The requirements for the terminal differentiation process of the stalk pathway (from prestalk to stalk) of Dictyostelium discoideum were analyzed by using a low-cell-density culture system with prestalk cells isolated from normally developed slugs. Any of the substances, such as differentiation-inducing factor-1 (DIF-1), DIF-2, dimethyloxazolidinedione, and adenosine, that had been shown to promote prestalk/stalk differentiation did not cause an efficient and consistent induction of prestalk-to-stalk conversion, either alone or in combination. However, the addition of 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP) resulted in high efficiency (80-90%) of stalk cell formation which accompanied the accumulation of a stalk-specific protein, wst34. The maturation process was inhibited by Ca2+ but not by Mg2+. More importantly, the Br-cAMP-induced stalk cell differentiation was neither inhibited nor promoted by DIF-1, cAMP, or ammonia and occurred even at very low cell densities if only Br-cAMP was supplied. Since Br-cAMP is though to penetrate into the cells and to activate the intracellular protein kinase A, the present results suggest that the activation of protein kinase A is sufficient for prestalk-to-stalk conversion.