xtracellular ATP modulates cardiac contraction through P2-purinoceptors on cardiac myocytes. To elucidate the molecular mechanism of this response, we examined the effects of P2-purinoceptor activation on phosphoinositide (PI) hydrolysis and the cAMP system in cultured ventricular myocytes of fetal mice. In a concentration-dependent manner, ATP stimulated accumulations of [3H]inositol monophosphate, bisphosphate, and trisphosphate with the half-maximum effective concentration of ∼1 μM in the myocytes labeled with [3H] inositol. The order of efficacy of a series of adenyl compounds for stimulation of PI hydrolysis was adenosine 5′-O-(3-thiotriphosphate) (ATPγS), ATP>ADP, 5′-adenylylimidodiphosphate (APPNP) > α,β-methyleneadenosine 5′-triphosphate (APCPP) > β,γ-methyleneadenosine 5′-triphosphate, AMP > adenosine. On the other hand, 100 μM ATPγS inhibited isoproterenol-induced accumulation of cAMP by ∼70% without decreasing the time to maximal cAMP levels, as measured by radioimmunoassay. This response was also concentration dependent, with a half-maximum inhibitory concentration (IC50) of ∼1 μM. All of the tested ATP, ADP, and ATP analogues inhibited the cAMP system, and the responses to ATPγS, APPNP, and APCPP were insensitive to an A1-purinoceptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. Pertussis toxin attenuated the ATP-induced PI hydrolysis by no more than 25% at 100 ng/ml but completely suppressed the ATPγS-induced inhibition of the cAMP system. Protein kinase C-activating phorbol ester, 4β-phorbol 12β-myristate 13α-acetate, inhibited the ATP-induced PI hydrolysis with an IC50of ∼1 nM and also attenuated the ATPγS-induced inhibition of the cAMP system at ≥1 nM, although a biologically inactive phorbol ester, 4α-phorbol 12,13 -didecanoate, did not. From these data, P2-purinoceptor activation stimulates PI hydrolysis by activating phospholipase C primarily through pertussis toxin-insensitive G proteins and attenuates cAMP accumulation by inhibiting adenylate cyclase through pertussis toxin-sensitive G proteins. Protein kinase C is likely to negatively regulate both the signal transduction systems. (Circulation Research1992;70:477–485)