OBJECTIVE To study the changes in nitric oxide synthase activities in human myometrium and trophoblast throughout pregnancy and around delivery. METHODS Samples of villous trophoblast were collected from women undergoing elective cesarean delivery at term (n = 12) or voluntary termination of pregnancy in the first (n = 27) or second (n = 11) trimesters of pregnancy. Myometrial samples were obtained from nonpregnant women undergoing hysterectomy (n = 5) and pregnant women both before (n = 7) and after (n = 7) the onset of spontaneous labor at term. Nitric oxide synthase activity was quantified for homogenized samples using the L-citrulline assay in the presence and absence of calcium. RESULTS The highest levels of nitric oxide synthase activity were found in first-trimester villi (range 2-29 nmol L-citrulline/minute/g protein), with a significant fall in activity in the third trimester (range 2-10 nmol L-citrulline/minute/g protein; P < .001 for both calcium-dependent and calcium-independent activity). Myometrial activities were relatively low compared with those in the trophoblast (0-2 nmol L-citrulline/minute/g protein), with no significant differences in calcium-dependent activities between subgroups. Myometrial calcium-independent activities were lower in pregnant than in nonpregnant women (P = .007), with those in labor having levels higher than those not in labor (P = .048). CONCLUSIONS Levels of nitric oxide synthase activity are relatively high in villous trophoblast, particularly during the first trimester. Although the contribution to total nitric oxide production in the uterus by myometrial nitric oxide synthase appears to be relatively small, nitric oxide produced by the trophoblast may play a role in maintaining uterine quiescence by a paracrine effect. Further work is needed to test this hypothesis and explore other possible roles for trophoblast-derived nitric oxide in early pregnancy.