The aim of this study was to characterize the effect of estrogen on the expression of neuronal and endothelial isoforms of nitric oxide (NO) synthase (NOS) in myometrium, endometrium, and caruncle (nonglandular endometrium) in nonpregnant sheep. Twenty sheep were castrated during synchronized estrus (Days 14–16) and 4 days after surgery treated i.v. through the jugular with 100 μg/day of estradiol-17β for 5 (n = 6) or 8 (n = 6) days or with vehicle (n = 8). Nitric oxide synthase mRNA was measured by ribonuclease protection assay, and NOS protein mass was measured by Western immunoblotting. Data were analyzed by ANOVA and Tukey's test. The three distinct uterine compartments studied contained the mRNA and protein for the neuronal (type I NOS) and the endothelial (type III NOS) isoforms of NOS. However, no inducible NOS was detected. Estrogen exhibited a differential effect on NOS expression in a tissue compartment- and NOS isoform-specific manner. In myometrium and caruncles, but not in endometrium, type I NOS mRNA and protein mass increased significantly (p < 0.05) after 5 or 8 days of estrogen. In contrast, type III NOS increased significantly in myometrium only after 8 days, whereas in endometrium and caruncles the increase was significant in the 5-day treatment group (p < 0.05). We conclude that the expression of type I NOS and type III NOS in the uterus are differentially regulated by estrogen. This differential regulation suggests that the NO produced within the uterus serves more than one physiological role. In myometrium it may be a uterorelaxant and regulate glucose utilization, and in endometrium and myometrium it may regulate blood flow.