We examined if rat myometrial cells in culture generate nitric oxide (NO) and express various isoforms of NO synthase (NOS). Myometrial cells isolated from rats on day 18 of gestation were incubated with various stimulators and inhibitors of NOS for 24 and 48 h, and NO production was evaluated by measuring nitrites in the media and NOS proteins in the cell lysates. NO was produced by myometrial cells and its production inhibited by N(G)-methyl-L-arginine (L-NMMA). This inhibition was reversed by L-arginine (3 mM). Interleukin-1beta (IL-1beta) significantly stimulated NO production, in a dose-dependent manner. The IL-1beta-stimulated NO production was inhibited by the NOS inhibitor, L-NMMA, whose effects were reversed by L-arginine. Abundant NOS III protein was detectable in freshly isolated myometrial cells, and this was maintained in culture in the presence of fetal bovine serum (FBS; 10%). In the absence of FBS, NOS III levels decreased significantly (by 90%) within 24 h. In contrast, NOS I and NOS II proteins were undetectable in freshly isolated muscle cells and in cells cultured without IL-1beta. However, NOS II protein in these cells was induced by IL-1beta. Thus, NO is produced by myometrial cells through the NOS III isoform, and the myometrial NO may be important in maintaining uterine quiescence during pregnancy.