BACKGROUND The purpose of this study was to compare in vitro contractile effects of oxytocin and carbetocin on human term pregnant myometrium with and without oxytocin pretreatment. METHODS This laboratory investigation was conducted on myometrial samples from women undergoing elective cesarean deliveries. The samples were dissected into four strips and suspended in individual organ bath chambers containing physiologic salt solution. After equilibration, they were pretreated with oxytocin 10 M (experimental group) or physiologic salt solution (control group) for 2 h and then subjected to dose-response testing with increasing concentrations of oxytocin or carbetocin (10 to 10 M). The amplitude, frequency, motility index (amplitude × frequency), and area under the curve of contractions were recorded and analyzed during the equilibration and dose-response periods. Comparisons were made between oxytocin-induced and carbetocin-induced contractions in control and oxytocin-pretreated groups. Motility index was the primary outcome measure. RESULTS Sixty-three experiments were performed (carbetocin, n = 31; oxytocin, n = 32) on samples from 18 women. The motility index of contractions (√g.contractions/10 min) produced by oxytocin was significantly higher than carbetocin in both control (regression-estimated difference, 0.857; 95% CI, 0.290 to 1.425; P = 0.003) and oxytocin-pretreated (0.813; 0.328 to 1.299; P = 0.001) groups. The motility index was significantly lower in oxytocin-pretreated groups than their respective controls for both oxytocin (-1.040; -1.998 to -0.082; P = 0.03) and carbetocin (-0.996; -1.392 to -0.560; P < 0.001). CONCLUSIONS In vitro contractions produced by oxytocin are superior to carbetocin in human myometrium with or without oxytocin pretreatment. Oxytocin pretreatment results in attenuation of contractions induced by both oxytocin and carbetocin.