Objective: We studied the effects of intracellularly applied inositol-1,4,5-trisphosphate (InsP 3 ) to test the hypothesis that InsP 3 is a messenger for stimulation of L-type calcium current (I C a ( L ) ) and contractions by muscarinic agonists. Methods: Voltage clamp pulses elicited I C a ( L ) that evoked contractions recorded with an edge detector in single guinea pig ventricular myocytes superfused with Tyrode's solution (36 o C). InsP 3 or cyclic AMP (cAMP) was dialyzed into the cell at selected times via the patch electrode. Results: InsP 3 (1-10 μM) transiently increased isotonic contractions when applied for 4-5 min; higher concentrations (50-300 μM) caused a sustained decrease in contractions. InsP 3 had no effect on I C a ( L ) at any concentration tested. Caffeine (10 mM)-induced contractures were increased and decreased, respectively, at 3 and 100 μM InsP 3 . Pentosan polysulfate (50 μg/ml), an InsP 3 receptor antagonist, opposed the increased contractions by InsP 3 . Intrapipette cyclic AMP (10-300 μM) caused sustained increases of I C a ( L ) and contractions. Cyclic AMP, but not InsP 3 , also increased I C a ( L ) when intrapipette Cs + suppressed K + currents. Conclusions: Increased myocyte shortening at low InsP 3 concentrations accords with receptor-initiated sarcoplasmic reticulum Ca 2 + release. The transient stimulation of contractions at low concentrations and the sustained reduction of contractions at high concentrations are not consistent with a role for InsP 3 in the persistent increase of contractions by muscarinic agonist in ventricular muscle and myocytes. The failure of InsP 3 to change I C a ( L ) when contractions were increased or decreased militates against the L-type calcium channel being an effector of InsP 3 .