OBJECTIVE To determine whether nitric oxide synthase isoforms are expressed in human myometrium, to define their localization, and to examine the possibility that one or more may play a role in initiating labor. METHODS Human myometrial biopsies were collected from term laboring and nonlaboring patients. The mRNA expression was assessed using in situ hybridization. The relative expression levels were also assessed by Affymetrix GeneChip hybridization. The protein expression was determined using Western blot analysis, and localization was shown using immunostaining. RESULTS Both inducible nitric oxide synthase and brain nitric oxide synthase were expressed in myometrial cells and blood vessel endothelial cells. Endothelial nitric oxide synthase was expressed exclusively in blood vessel endothelial cells. There were no differences in distribution or quantity of nitric oxide synthase expression between term labor and nonlabor myometrium. CONCLUSIONS All three isotypes of nitric oxide synthases were expressed in human pregnant term uterus. Their expression did not change in the myometrium of the laboring uterus.