Recent biomolecular studies have shown that continuous exposure of human myometrial cells to oxytocin results in a significant loss of responsiveness to subsequent oxytocin stimulation, perhaps because of desensitization of the oxytocin receptors. However, it is unclear whether this phenomenon results in a reduction of the contractile activity of the uterine muscle in humans or animals. The objective of our study was to investigate the in vitro response of the uterine muscle of pregnant rats to oxytocin, following preexposure to varying concentrations of oxytocin, for varying durations. Longitudinal myometrial strips were isolated from 16 pregnant Wistar rats at 19 to 21 days of gestation and preexposed to oxytocin 10(-10) or 10(-8) mol/L (experimental groups) or physiological salt solution (control groups) for 1- or 4-hour period. All muscle strips were then subjected to a dose-response study with oxytocin 10(-10) to 10(-5) mol/L. The area under the curve, frequency, and amplitude of contractions were recorded and compared between the groups. The area under the curve, frequency, and amplitude of the oxytocin-induced contractions were all significantly suppressed in the groups preexposed to oxytocin 10(-8) mol/L compared to either the control groups (P < .0001) or the groups preexposed to oxytocin 10(-10) mol/L (P < .0001). There was no difference in the oxytocin-induced myometrial contractions between the groups preexposed to oxytocin for either the 1- or 4-hour periods. The inhibition of the oxytocin-induced contractile response of pregnant rat myometrium is observed as early as 1 hour of preexposure to oxytocin and is dependent on the preexposed oxytocin concentration and not on the duration of its exposure.