Prolonged continuous oxytocin administration during labor may induce oxytocin receptor desensitization, which attenuates the response of the myometrium to further oxytocin, increasing the risk of postpartum hemorrhage. The literature comparing pulsatile (intermittent) versus continuous oxytocin administration for induction and augmentation of labor is inconsistent with regard to maternal outcomes. We aimed to determine the effect of intermittent versus continuous oxytocin preexposure on myometrial responsiveness to subsequent oxytocin. We hypothesized that intermittent oxytocin pretreatment would result in superior subsequent oxytocin-induced contractility than continuous oxytocin pretreatment. This in vitro study was undertaken using myometrium obtained from women undergoing elective cesarean deliveries. Each myometrial strip was mounted in an individual organ bath with physiological salt solution under homeostatic conditions and allocated to one of 3 groups: (1) control (no pretreatment); (2) continuous (pretreatment with oxytocin 10(-5) M for 2 hours); or (3) intermittent (pretreatment with alternating oxytocin 10 M and physiological salt solution every 15 minutes, for 2 hours). After pretreatment, dose-response testing to oxytocin 10(-5) to 10(-5) M was performed and contractile parameters were measured. The primary outcome was motility index (MI, amplitude × frequency) of contractions. Eighteen women were recruited, and 86 successful experiments were performed (control n = 29, continuous n = 28, intermittent n = 29). The means (standard errors) of MI (√g·contractions/10 min) in the control, continuous, and intermittent groups were 2.34 (0.09), 1.78 (0.09), and 2.13 (0.11), respectively. The MI was significantly reduced in the continuous group when compared to the control (estimated difference [95% confidence interval {CI}], -0.56 [-0.81 to -0.31]; P < .01) and intermittent group (estimated difference [95% CI], -0.35 [-0.62 to -0.08]; P = .01). There was no significant difference in MI between the intermittent and control group (estimated difference [95% CI], -0.21 [-0.51 to 0.09]; P = .17). Human myometrium remains more responsive to subsequent oxytocin after intermittent compared to continuous exposure to oxytocin, most likely due to reduction in oxytocin receptor desensitization, or facilitation of receptor resensitization in the intermittent group. Hence, intermittent oxytocin administration during labor warrants further investigation as a technique to preserve uterine oxytocin responsiveness.