Human mammary carcinoma cells (MCF-7) were arrested in late G1-phase after treatment with agents (forskolin, interleukin-1 beta 3-isobutyl-1-methylxanthine) that increased the endogenous concentrations of cAMP. The effect of elevated cAMP was mimicked by microinjected catalytic (C alpha) cAMP-dependent protein kinase (cAK) subunit and reversed by the injection of a dominant negative cAK regulatory mutant (RID199). Further evidence that activation of cAK induced growth arrest was provided by the use of pairs of stable cAMP analogs known to synergistically activate isolated cAK isozymes. Furthermore, the effect of cAMP was not potentiated by serine/threonine phosphatase inhibitors that profoundly restricted MCF-7 growth. Some 8-substituted cAMP analogs, e.g. 8-Cl-cAMP and 8-NH2-cAMP, induced cell death rather than reversible inhibition of growth. Their effect was not synergized with complementary cAMP analogs. Furthermore, their potency was decreased rather than increased in the presence of an inhibitor of degradation (3-isobutyl-1-methylxanthine). Finally, their effect could be mimicked by degradation products unable to activate cAK. We concluded that 8-Cl-cAMP (and 8-NH2-cAMP) induced irreversible growth arrest by a mechanism not involving cAK, whereas activation of cAK resulted in a transient and fully reversible inhibition of cell proliferation.