Oestradiol can stimulate osteoblast activity. Osteoblast function is thought to be regulated by nitric oxide (NO), We hypothesised that the effect of 17 beta -oestradiol (17 beta -E-2) on osteoblast activity is mediated by NO. This hypothesis was tested using osteoblasts isolated from human trabecular bone, calvariae of rats, endothelial NO synthase (eNOS) gene-deficient mice, and their wild-type counterparts. Our results show that 17 beta -E-2 dose-dependently stimulated proliferation and differentiation of primary human, rat and wild-type osteoblasts. The presence of N-G-monomethyl-L-arginine (10(-3) M), an inhibitor of NOS activity, blocked the 17 beta -E-2-(10(-7) M)-induced increases in thymidine incorporation (P < 0.01), alkaline phosphatase activity (P < 0.01) and bone nodule formation (P < 0.01) of wild-type, human and rat osteoblasts, respectively. Moreover, 17-E-2 did not induce a response in eNOS gene-deficient osteoblasts. 17 beta -E-2 also increased total eNOS enzyme expression in rat osteoblasts. These findings indicate 17 beta -E-2 modulates osteoblast function by NO-dependent mechanisms mediated via the eNOS isoform,