The effects of the uterine relaxants relaxin and isoproterenol on intracellular free calcium and inositol phosphate formation were investigated in rat myometrium. Preincubation of fura-2-loaded myometrial cell suspensions with relaxin and isoproterenol inhibited the oxytocin-induced stimulation of intracellular free calcium, with EC50 values of 0.02 and 1.0 .mu.M, respectively. Pretreatment of cells with pertussis toxin or replacement of extracellular calcium with 2 mM EGTA inhibited the oxytocin-induced increase in intracellular calcium by 47% and 50%, respectively, but did not inhibit the action of relaxin. (Bu)2cAMP and forskolin also inhibited the effect of oxytocin on intracellular calcium. In uterine strips prelabeled with [3H]inositol, oxytocin stimulated a dose-dependent accumulation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate, with an EC50 of 0.38 .mu.M, and pertussis toxin inhibited this effect. Relaxin, isoproterenol, chlorophenylthio-cAMP, and forskolin inhibited the oxytoxin-stimulated formation of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. The effect of relaxin on inositol monophosphate, inositol bisphosphate, and inositol triphosphate. The effect of relaxin on inositol trisphosphate formation was dose dependent, with an EC50 for 0.1 .mu.M. Relaxin and isoproterenol also inhibited inositol phosphate formation in myometrial cells. These data demonstrate the attenuation of contractant-induced elevation in myometrial intracellular calcium and phosphoinositide turnover by uterine relaxants and suggest that these actions may be related. In addition, they provide additional evidence that cAMP-mediated mechanisms may be involved in mediating uterine relaxation.