Glutathione (GSH) is required for the oxidation of formaldehyde (HCHO) to formate catalyzed by formaldehyde dehydrogenase (FDH). The effects of GSH depletion on the mechanisms of labeling of macromolecules in the rat nasal mucosa and bone marrow by 3HCHO and H14CHO were investigated. Male rats were exposed for 3 hr to atmospheres containing 3HCHO and H14CHO at concentrations of 0.9, 2, 4, 6, or 10 ppm, 1 day after a single 3-hr preexposure to the same concentration of unlabeled HCHO. Two hours prior to the second exposure, the animals were injected either with phorone (300 mg/kg, ip) or with corn oil. The concentration of nonprotein sulfhydryls in the nasal respiratory mucosa of phorone-injected rats was decreased to 10% of that of corn oil-injected rats. The metabolic incorporation of 3HCHO and H14CHO into DNA, RNA, and proteins in the respiratory and olfactory mucosa and bone marrow (femur) was significantly decreased, and DNA-protein crosslinking was significantly increased in the respiratory mucosa of phorone-injected relative to corn oil-injected rats at all HCHO concentrations. DNA-protein crosslinks were not detected in the respiratory mucosa of corn oil-injected rats at 0.9 ppm. Evidence was obtained for the formation of adducts of HCHO with the RNA from the nasal respiratory mucosa of phorone-injected rats at concentrations above 0.9 ppm. Covalent binding of HCHO to macromolecules in the bone marrow was not detected. These results indicate that the GSH-dependent oxidation of HCHO catalyzed by FDH is an important defense mechanism against the covalent reactions of HCHO with nucleic acids in the respiratory mucosa. Experiments using phorone-injected rats exposed to 10 ppm of [3H]- and [14C]formaldehyde showed that the DNA from the respiratory mucosa was enriched in 3H relative to 14C in comparison to the inhaled vapor. The enrichment is explained by an isotope effect in the oxidation of 3HCHO and H14CHO (H. d'A, Heck and M. Casanova (1987). Toxicol. Appl. Pharmacol. 89, 122-134), which results in 3H enrichment of the residual (unoxidized) HCHO that binds to DNA. A non-linear pharmacokinetic model is proposed that depicts the potential effects of FDH saturation on the relative concentrations of intracellular to extracellular HCHO.