Inducible nitric oxide synthase (iNOS) mRNA is up-regulated in vivo by dibutyryl-cAMP (db-cAMP), the purine-2y receptor agonist 2-methylthio-ATP and Escherichia coli endotoxin lipopolysaccharide (LPS). Ethanol and diethyldithiocarbamate inhibit LPS-stimulated iNOS mRNA. Their effects on db-cAMP- and 2-methylthio-ATP-stimulated iNOS mRNA remain undefined. We examined the effect of ethanol (4.5 g/kg intraperitoneal) and intratracheal diethyldithiocarbamate (5 mg/kg) on intratracheal LPS (0.6 mg/kg), db-cAMP (0.1 and 1 mg/kg) or 2-methylthio-ATP (5 mg/kg)-stimulated rat alveolar macrophage (AM) iNOS mRNA and protein, reactive nitrogen intermediates nitrite and nitrate anion (RNI) and nuclear transcription factor-kappaB (NF-kappaB) in vivo. LPS and the autacoids increased iNOS mRNA and protein in rat AM and RNI in bronchoalveolar lavage fluid and in ex vivo incubates of AM compared with these parameters in control rats (n = 6-21/group). Only LPS up-regulated TNF-alpha mRNA and release of TNF-alpha in bronchoalveolar lavage fluid and AM. Ethanol inhibited LPS stimulation of the iNOS cascade at the level of transcription but inhibited only autacoid-stimulated iNOS protein and RNI. Diethyldithiocarbamate selectively inhibited the LPS-stimulated iNOS cascade at the level of transcription. Coadministration of ethanol and diethyldithiocarbamate inhibited LPS-stimulated iNOS mRNA, protein and RNI more than either inhibitor alone but did not differ from ethanol alone on autacoid-stimulated iNOS protein or RNI. LPS increased and db-cAMP did not affect NF-kappaB in AM. Ethanol inhibited LPS-stimulated NF-kappaB. Thus, two distinct pathways exist for induction of iNOS mRNA in rat AM in vivo: an NF-kappaB pathway for LPS and cytokines inhibitable by ethanol and diethyldithiocarbamate and an NF-kappaB-independent pathway, refractory to inhibition by ethanol and diethyldithiocarbamate for db-cAMP and 2-mes-ATP. Finally, ethanol inhibits iNOS at the level of transcription and at the level of the enzyme.