The influence of isoproterenol (10(-6) M) and 3-isobutyl-1-methylxanthine (IBMX) (10(-6) M) on the junctional conductance (gj) of isolated rat ventricular cell pairs was investigated. It was found that both drugs increased gj within 25-30 s. To test the phosphorylation hypothesis, cAMP-dependent protein kinase inhibitor (20 micrograms/ml) was dialyzed into both cells of the pair. The protein kinase inhibitor suppressed the effect of both drugs on gj. Moreover, the protein kinase inhibitor by itself, reduced gj by 18% (S.E. +/- 9) (n = 10), suggesting that basal levels of cAMP in the cells contributes to modulation of gj. Dialysis of cAMP-protein kinase inhibitor (20 micrograms/ml) in just one cell of the pair induced rectification of the junctional membrane. These results indicate that the activation of cAMP-dependent protein kinase is necessary for the effect of cAMP on junctional conductance in heart.