Previous studies have shown evidence of constitutive and cytokine-inducible nitric oxide (NO) synthase activity in cultured osteoblast-like cells from various species. Although cytokine-induced NO production has been found to inhibit osteoblast growth, the role of constitutive NO production in regulating osteoblast function is less clear and the isoforms of nitric oxide synthase (NOS) that are expressed by human osteoblasts have not been determined. Here, we investigated NOS expression in cultured human osteoblast-like cells and studied the effects of constitutive and cytokine-induced NO on osteoblast growth and differentiation. Low levels of NO were produced constitutively by osteoblast-like cells as reflected by analysis of medium nitrite concentrations, and evidence of ecNOS mRNA, protein, and bioactivity was found in primary osteoblasts (hOBs), TE85, and MG63 osteosarcoma cells. None of the osteoblast-like cells expressed nNOS, however, and iNOS was produced only by hOB cells after stimulation with the cytokines IL-1beta, TNF-alpha, and IFN-gamma. The NOS inhibitor, L-NMMA, did not affect growth or alkaline phosphatase activity in unstimulated osteoblasts. Incubation of hOB cells with cytokines inhibited growth and stimulated alkaline phosphatase activity and these effects were abrogated by L-NMMA. Cytokines also inhibited growth of TE85 cells and MG63 cells, but these effects appeared to be NO independent because they were not influenced by L-NMMA. Our experiments show that human osteoblasts constitutively produce NO through the ecNOS pathway, but demonstrate that this does not appear to exert an appreciable effect on osteoblast growth or differentiation under basal conditions. In contrast, IL-1beta, TNF-alpha, and IFN-gamma exerted growth-inhibiting and differentiation-inducing effects on osteoblasts that were partly NO dependent, indicating that NO may act predominantly as a modulator of cytokine-induced effects on osteoblast function.