Acetylcholine (ACh) causes early activation events in mouse oocytes, but little is known about its precise role in the early embryonic development of mice. We aimed to determine whether and how ACh is capable of rescuing two-cell block in an in vitro culture system. ACh evoked different transient Ca(2+) patterns showing a higher Ca(2+) peak in the two-cell stage embryos (two-cells) than observed in mature oocytes. In early two-cells subjected to an in vitro two-cell block, xestospongin C (Xes-C), an IP3 receptor antagonist, significantly decreased the level of the ACh-induced Ca(2+) increase. The reduction in the ACh-induced Ca(2+) increase by Xes-C in late two-cells was lower than that in early two-cells. Furthermore, KN62 and KN93, both CaMKII inhibitors, were found to reduce the magnitude of the ACh-induced Ca(2+) increase in early two-cells. The addition of ACh to the culture medium showed an ability to rescue in vitro two-cell block. However, the addition of ACh together with both Xes-C and CaMKII inhibitors or with either inhibitor separately had no effect on the rescue of two-cell block. Long-term exposure of late two-cells to ACh decreased morula and early blastocyst development and ACh had a differential effect on early and late two-cells. These results indicate that ACh likely rescues the in vitro two-cell block through activation of IP3R- and/or CaMKII-dependent signal transduction pathways.