Inositol-1,4,5-trisphosphate (IP3) has been proposed to be a second messenger in response to alpha-l-adrenoceptor stimulation also in myocardial cells. We studied the effect of alpha-l-adrenoceptor stimulation (5 x 10 -5 tool/1 phenylephrine or 5 x 10 -5 mol/1 noradrenaline both in the presence of 10 4 tool/1 timolol) on IP 3 mass content in isolated perfused rat hearts. IP 3 content was determined by a specific receptor-binding assay-kit (TRK 1000, Amersham) after validating the method. For comparison also the effect ofmuscarinic stimulation (10 4 mol/1 carbachol in the presence of 104 mol/1 timolol) on IP 3 content was measured in corresponding preparations. A basal IP 3 level of about 75 pmol/mg protein was found. There were no prominent effects of alpha-1-adrenoceptor stimulation on total IP 3 content in isolated perfused rat hearts. Phenylephrine gave a statistically significant increase of about 40% at 1/4 min and a statistically significant decrease of about 25% at 4 min after start of exposure. Noradrenaline, however, gave no statistically significant change ofIP 3 at the time-points studied. Muscarinic stimulation caused a slight, statistically insignificant, increase ofIP 3 at 1/4 min. The results are compatible with an assumption that agonist stimulation evokes a localized increase ofIP 3 which may be masked by a relatively high total IP 3 mass content. The IP 3 peak after phenylephrine coincided with the early positive inotropie phase of the response reported earlier in perfused rat hearts for alpha-1-adrenoceptor stimulation by phenylephrine. Although this might be compatible with a role for IP 3 in this early and transient phase, a mediator function ofIP 3 in the inotropic response is not established. (Mol Cell Biochem 163/164: 167-172, 1996)