Oxytocin pretreatment of pregnant rat myometrium has been shown to reduce the contractions produced by further administration of oxytocin, as a function of the desensitization phenomenon. It is unclear whether this phenomenon affects the contractions produced by various prostaglandins that are used in the management of postpartum hemorrhage. The objective of this study was to investigate the contractile effects of various prostaglandins after oxytocin pretreatment and to compare their relative efficacies in vitro on pregnant rat myometrial strips. Myometrial samples from 29 pregnant Wistar rats at term were isolated and pretreated with oxytocin (10(-8) mol/L, experimental group) or physiological salt solution (control group) for 1 hour. They were then subjected to dose-response testing with oxytocin (n = 32), PGF2α (n = 16), dinoprostone (n = 14), alprostadil (n = 14), or misoprostol (n = 15) with cumulative increases in the organ bath concentrations from 10(-10) to 10(-5) mol/L. The contractile efficacies of various prostaglandins and oxytocin during the dose response were analyzed using mixed linear modeling and compared between the groups. There was no significant difference in the amplitude, frequency, motility index (amplitude × frequency), or area under the curve of all prostaglandins between the groups pretreated with oxytocin and the control group. However, there was a significant decrease in the frequency (P = .02) and motility index (P = .05) in the dose-response curves of oxytocin in the groups pretreated with oxytocin compared with the control groups. Overall, oxytocin produced superior contractions compared with all other prostaglandins, while dinoprostone and misoprostol produced the weakest contractions. The uterotonic effects of various prostaglandins are not affected by oxytocin desensitization; and despite desensitization, oxytocin provides superior contractions compared with the prostaglandins.