To test the hypothesis that binding site regulation is the primary process controlling the responsiveness of the rat's myometrium to oxytocin during pregnancy, I have studied the effects of oxytocin on longitudinal and circular strips of myometrium in vitro throughout pregnancy. Longitudinal muscle was as sensitive on day 10 of pregnancy (EC50 = 1.6 nM) as it was at term (EC50 = 1.3 nM) and there was no significant change in the mean maximal force developed in response to the hormone (2.1 ± 0.9 vs. 1.5 ± 0.3 N cm−2). Circular muscle on the other hand was essentially refractory to the hormone until day 21 of pregnancy at which time its sensitivity and the maximum response were similar to those of longitudinal muscle. These results indicated that regulation of oxytocin sensitivity in the two muscle layers was temporally different, and they suggested different mechanisms. The effect of oxytocin on longitudinal muscle was not compatible with the hypothesis that changes in binding site number regulate the responsiveness of the tissue, whereas the effect on circular muscle was.