Membranes of cultured newborn rat cardiomyocytes contain enzymatic activities that regulate the formation and the breakdown of inositol 1,4,5-trisphosphate (1,4,5-IP3). GTP gamma S increased the rate of exogenous [3H]phosphatidyl 4,5-bisphosphate ([3H]PIP2) hydrolysis (EC50: 40 microM). This effect was dependent on the presence of deoxycholate and maximal at 2 mM deoxycholate. GTP gamma S increased the efficacy of phospholipase C (PLC) (by 2.3-fold), but did not alter the apparent affinity of the enzyme for PIP2. Other nucleotides, GDP beta S and ATP gamma S, and pyrophosphate also stimulated PIP2 hydrolysis, while AlF4- was ineffective. The effect of GTP gamma S was not inhibited by GDP beta S. The agonists norepinephrine and thrombin, which by themselves had no effect, did not potentiate the response to GTP gamma S. In contrast, 1,4,5-IP3 hydrolysis was decreased by GTP gamma S (EC50: 100 microM) as well as by other nucleotides and by pyrophosphate, but not by AlF4-. GDP beta S did not antagonize the GTP gamma S-induced inhibition of IP3 hydrolysis. These results suggest that GTP can stimulate the hydrolysis of exogenous PIP2 by an action on membrane-bound PLC at a site beyond the G protein activating PLC and inhibit the hydrolysis of 1,4,5-IP3 by a mechanism common to all nucleotides. Thus, GTP can regulate 1,4,5-IP3 metabolism by stimulating its formation and inhibiting its breakdown.