Nitric oxide (NO) acts as a regulator in cell proliferation and expression of growth factors and forms peroxynitrite (ONOO(-)) in oxidative conditions. The aim of the study was to investigate the role of NO in cellular response to hyperbaric oxygen (HBO). NO and nitrotyrosine (NT), biochemical marker for ONOO(-), cell proliferation and growth factors, were ex-vivo studied in cell cultures under HBO and normobaric (NOR) conditions. A549 (epithelial), L929 (fibroblast) and SVEC (endothelial) were exposed to 100% O(2), at P = 280 kPa for t = 60 min, once daily for five sessions. Cell proliferation was determined as the incorporation of bromodeoxyuridine (BrdU) into cells and NO as nitrates/nitrites (NO(3) (-)/ NO(2) (-)) Gries reaction product in cell culture supernatant (CCSP). NT, vascular endothelial growth factor (VEGF) and transforming growth factor-beta 1 (TGFb1) were measured with enzyme-inked immunosorbent assay (ELISA) in CCSP. The time course of total NO was opposite to that of cell proliferation in HBO conditions, peaking after the second HBO session, while cell proliferation showed a reverse trend, minimizing at the same time, suggesting a reverse and transient anti-proliferative effect. Released growth factors were significantly increased in late HBO sessions. NT peaked after second treatment, indicating the formation of ONOO(-). In control cultures (NOR), proliferation rate was downward and no significant differences were found for the other parameters. In conclusion, the data suggested a key role for NO in the beneficial HBO action, depending on its concentration, which fluctuated with the time of HBO exposure and the activation of oxidant-antioxidant (REDOX) mechanisms, regardless of cell type.