1. We studied the effects of ovalbumin (OA) sensitization and challenge on inducible nitric oxide synthase (iNOS) gene and protein expression in Brown-Norway rats in vivo. 2. By use of Northern analysis, a 4.4-kb iNOS mRNA transcript was weakly observed in control rat lung but there was a 3 fold increase in lungs sensitized to OA alone (P<0.05). In sensitized rats, four hours after exposure to OA aerosol, there was a 6 fold increase in iNOS mRNA transcript (P<0.05), which returned to baseline at 24 h. 3. Immunostaining with an anti-mouse iNOS antibody revealed some patchy staining of airway epithelium in naive rats. There were no changes in sensitized rats exposed to saline, but sensitized and OA-exposed rats showed increased expression in iNOS staining in macrophages. 4. Electrophoretic mobility shift assays of lung nuclear extracts showed a marked increase in nuclear factor-kappaB (NF-kappaB)-binding activity at 2 h after allergen exposure with return to baseline at 6, 12 and 24 h. 5. We concluded that there is increased iNOS gene and protein expression associated with increased NF-kappaB DNA-binding in lungs of sensitized and challenged rats. The increase in iNOS expression may underlie the increase in exhaled NO found after allergen challenge and may contribute to the development of allergen-induced airway hyperresponsiveness.