Oxytocin (OT) receptors are important regulators of myometrial contractility. By using the activity of large conductance Ca²⁺-activated K⁺ (BK_Ca) channels as readout, we analyzed OT signaling in cells from non-pregnant (NPM) and pregnant (PM) rat myometrium in detail. In nystatin-perforated whole-cell patches (NPP) from NPM cells which leave the intracellular integrity intact, OT transiently increased BK_Ca mediated outward currents (I_out). This OT-evoked I_out was caused by the Ca²⁺ transients in response to the G_q/11-mediated activation of phospholipase C (PLC) and was inhibited by activation of protein kinase A (PKA). In an open-access whole-cell patch (OAP), the OT-induced transient rise in I_out was disrupted whereas the regulation of BK_Ca by the cAMP/PKA cascade remained intact. OT counteracted the isoprenaline, thus -adrenoceptor/G_s-mediated effect in NPM cells measured in OAP. In contrast, OT further enhanced the -adrenoceptor/G_s-mediated effect on BK_Ca activity in PM cells. All OT-effects in the OAP were mediated by pertussis toxin (PTX) sensitive G_i proteins and PKA. By quantitative real-time PCR and over-expression of the recombinant protein we demonstrate that an upregulation of the G-stimulated adenylyl cyclase (AC) II during pregnancy is most likely responsible for this switch. By studying the OT-evoked I_out in NPP of PM cells, we further detected that the OT receptor/G_i mediated co-activation of AC II enhanced the -adrenoceptor/G_s induced suppression of the OT-evoked Ca²⁺ transients and thus diminishes and self-limits OT-induced contractility. The differential regulation of the PKA-mediated suppression of OT-evoked Ca²⁺ transients and BK_Ca activity likely supports uterine quiescence during pregnancy.