The sensory neurons of the olfactory epithelium are continually renewed during the entire lifespan in mammals. The stem cells responsible for cellular replacement are among the basal cells of this epithelium (Mackay-Sim & Kittel, 1991). The enzyme nitric oxide synthase (NOS), which generates the diffusible messenger nitric oxide (NO), is transiently expressed during neurogenesis in the olfactory bulb and in the regenerating olfactory epithelium of adult animals (Roskams et al. 1994). To determine the role played by NO during olfactory neurogenesis, we used a serum-free system for culturing non-neuronal cells. Olfactory epithelia were obtained from adult rats sacrificed with an anaesthesia overdose (pentobarbitone 100 mg/Kg. ip). This protocol was approved by the ethics committee of all universities involved. We studied the presence of neuronal (nNOS), endothelial (eNOS) and inducible (iNOS) NOS isoforms, and the effects of NOS inhibitors and NO donors under conditions that promote proliferation or differentiation of olfactory epithelial cells in vitro. Proliferation and differentiation of the neuronal precursors were induced with fibroblast growth factor (FGF-2) and transforming growth factor (TGF-β2), respectively. We detected by immunocitchemistry the expression of the three enzyme isoforms under both conditions. Furthermore, we found that nNOS was present in tubulin βIII and neurofilament positive cells (immature neuronal markers) and in some GBC-1 positive cells (globose basal cells marker), but not in cytokeratin-14 positive cells (horizontal basal cells marker). In cultures treated with EGF/FGF-2 and TGF-β2, the proportion of cell in proliferation and differentiation determined by BrdU incorporation and neurofilament expression was 42.6±2.6% and 43.5±2.5%, respectively. The NOS inhibitors 1-2-trifluoromethylphenyl imidazole (TRIM) and L-N⁶-1-iminoethyl-lysine (N-NIL) reduced proliferation and increased differentiation in a dose-dependent manner. However, 10-100 μM N-NIL (K_i 10 times lower than TRIM for iNOS), showed a stronger anti-proliferative effect (13.6±3.1% and 4.0±1.4% of control, respectively; p<0.05) than TRIM at the same concentration (63.2±5.6% and 39.2±2.9%). 100 μM SNAP increased the proportion of cells in proliferation (57.1±3.3%, p<0.05). Besides, we found iNOS, but not nNOS nor eNOS expression in the regenerating olfactory epithelium. These results support the idea that NO produced by iNOS participates in neurogenesis, stimulating basal cell proliferation in the olfactory epithelium, independently of cGMP.