Phospholipase C (clostridium perfringens) significantly increased the cyclic nucleotide phosphodiesterase activity of a crude 105 000 g supernatant from rat heart. This activation only concerned the basal activity of cyclic GMP phosphodiesterase determined with 0.25 microM cyclic GMP as substrate, in the presence of EGTA, whereas stimulation was found to be independent of EGTA when phosphodiesterase activity was measured with 0.25 microM cyclic AMP. Similar qualitative results were found for the three cytosolic forms of phosphodiesterase separated from rat heart supernatant by isoelectric focusing. Supplementary experiments provided evidence that the activation of the cyclic AMP-specific phosphodiesterase was attributable to Phospholipase C activity and not to contaminating protease(s). In contrast, the stimulation of the cyclic GMP phosphodiesterase activity appeared to be largely dependent on the proteolytic activity of commercial Phospholipase C. Phosphatidic acid also significantly increased the cyclic AMP phosphodiesterase activity of the rat heart cytosol. These results suggest that the activation of cardiac cyclic AMP phosphodiesterase may be related to changes in phospholipid metabolism, notably the accumulation of phosphatidate, and relevant to physiological regulatory processes.