Changes in responsiveness of freshly isolated longitudinal muscle cells from rat uterus to oxytocin during gestation were investigated through measuring contractility as well as intracellular free calcium concentration. We have demonstrated the pregnant stage-dependent contraction of freshly isolated myometrial cells in response to an extracellular hormone, oxytocin, in Ca2+-containing medium. The oxytocin effect appeared to be through oxytocin receptor since the effect could be blocked by a specific oxytocin antagonist. The magnitude of the contraction of the isolated cells in response to extracellular oxytocin was in the order of 21 day >> 18 day > 15 day pregnant rat longitudinal muscle cells. In a concentration dependent manner, oxytocin elicited a rapid increase in [Ca2+]i of longitudinal muscle cells isolated from different stages of the pregnant rat uterus, especially at the term of pregnancy. The time (4-5 s) required to reach a maximum increase in [Ca2+]i of the isolated longitudinal muscle cells in response to oxytocin was the shortest among all previously reported studies. The results also indicated that the freshly prepared longitudinal muscle cells maintained their functional calcium signaling system. The order of the responsiveness of the isolated longitudinal muscle cells to oxytocin was 21 day >> 18 day > 15 day pregnant rats in terms of rate, affinity and magnitude. Oxytocin appears to transmit its signal mainly through stimulating a voltage-dependent and/or receptor operated nonselective calcium channel. However, the possibility that a part of the oxytocin action occurs through stimulating the release of calcium from intracellular store sites of longitudinal muscle still remains.