We determine whether the cyclic AMP signal transduction pathway affects phosphorylation of cyclic AMP response element binding protein and increases muscle gene expression in the heart. Elevation of cyclic AMP results in phosphorylation of the binding protein which is detected using an antibody specific for the phosphorylated, but not the unphosphorylated, form. The protein is present, but not phosphorylated, within the nuclei of myocytes in intact neonatal rat hearts and in high-density cultures. It is not expressed in low-density cultures. Increasing the amount of phosphorylated cyclic AMP with either isoproterenol or forskolin also increases the frequency and force of the beating. The phosphorylated form of the response element binding protein is visible in the nuclei by 10 min and persists for 2 h of drug treatment. A 1.5-fold increase in skeletal alpha-actin and alpha-myosin gene expression is detected after 48 h of isoproterenol treatment. However, blockage of beating with a calcium channel blocker (verapamil) in the presence of cyclic AMP results in a similar increased gene expression. This suggests that muscle gene expression can be regulated directly by the cyclic AMP pathway, probably via phosphorylation of the cyclic AMP response element binding protein but independent of contractile activity.