The mechanisms that mediate changes in uterine activity from a quiescent state during pregnancy to active labor at parturition are unknown. Nitric oxide (NO), a potent mediator of smooth muscle relaxation, and its presence in the uterus is the subject of this report. Nitric oxide synthase (NOS) activity was demonstrated in nerves, blood vessels and decidua of gravid rat uterus by the NADPH-diaphorase staining method. Uterine tissue fixed during labor demonstrated markedly less NOS. Quantitation of NOS activity in subcellular fractions of pregnant and laboring uterus revealed its presence in both the cytosolic and the membranous compartments of uterine homogenates. In both cellular subfractions the enzyme activity decreased significantly from pregnancy to term. We conclude NOS is present in multiple structures within the uterus. Its presence in two cellular compartments suggests more than one form of NOS may be present in the uterus. Reduction in NOS activity at parturition suggests NO may contribute to the maintenance of uterine contractile quiescence during gestation.