The effects of N6,O2-dibutyrl-adenosine-3',5'-cyclic monophosphate (db-cAMP) and 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP) on tension and cAMP-dependent protein kinase (PKA) activities in rat vas deferens were investigated. A soluble enzyme fraction obtained from the vas deferens was found to contain both type I and type II isozymes of PKA, whereas a particulate fraction contained only the type II isozyme. Exposure of the vas deferens to db-cAMP (1-100 microM) for 30 min caused a concentration-dependent inhibition of phenylephrine-induced contractions, with an EC50 of less than 10 microM. 8-Br-cAMP had no significant effect on contractions over a similar concentration range. Both of the analogs were able to activate PKA significantly at a concentration of 10 microM, and the magnitude of the PKA activation was greater with 8-Br-cAMP than with db-cAMP. Charcoal was added to the homogenization buffer in these experiments to prevent the artifactual activation of PKA by cAMP analogs trapped in the extracellular space. The ability of db-cAMP, but not 8-Br-cAMP, to inhibit the contraction of vas deferens could not be explained on the basis of differential activation of soluble or particulate PKA or of specific isozymes of the enzyme. It is, therefore, concluded that activation of PKA is not responsible for the relaxant effects of cAMP analogs in some smooth muscle.