Intracellular free Ca2+ concentration ([Ca2+]i) was monitored using the fluorescence from the dye Fura-2-AM in single myometrial cells from pregnant rats. Oxytocin and acetylcholine applied to the cell evoked an initial peak in [Ca2+]i followed by a smaller sustained rise which was rapidly terminated upon removal of acetylcholine or persisted after oxytocin removal. A Ca2+ channel blocker (oxodipine) and external Ca2+ removal decreased both the transient and sustained rises in [Ca2+]i suggesting that Ca2+ influx through L-type Ca2+ channels participated in the global Ca2+ response induced by oxytocin. However, the initial peak in [Ca2+]i produced by oxytocin was mainly due to Ca2+ store release: it was abolished by inclusion of heparin [which blocks inositol 1,4,5-trisphosphate (InsP3) receptors] in the pipette (whole-cell recording mode of patch-clamp) and external application of thapsigargin (which blocks sarcoplasmic reticulum Ca(2+)-ATPases). In contrast, the transient Ca2+ response induced by oxytocin was unaffected by ryanodine. Moreover, caffeine failed to induce a rise in [Ca2+]i but reduced the oxytocin-induced transient Ca2+ response. The later sustained rise in [Ca2+]i produced by oxytocin was due to the entry of Ca2+ into the cell as it was suppressed in external Ca(2+)-free solution. The Ca2+ entry pathway is permeable to Mn2+ ions, in contrast to that described in various vascular and visceral smooth muscle cells. Oxytocin-induced Ca2+ release is blocked by the oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT. The prolonged increase in [Ca2+]i after oxytocin removal is rapidly terminated by addition of the oxytocin antagonist suggesting that oxytocin dissociation from its receptor is very slow.(ABSTRACT TRUNCATED AT 250 WORDS)