The mechanisms of labeling of macromolecules (DNA, RNA, and protein) in the respiratory and olfactory mucosa, and in the bone marrow (femur) of male Fischer-344 rats exposed to [14C]- and [3H]formaldehyde [( 14C]- and [3H]CH2O) were investigated. Animals were exposed for 6 hr to atmospheres containing [14C]- and [3H]CH2O at concentrations of 0.3, 2, 6, 10, or 15 ppm, 1 day following a single pre-exposure to the same concentration of unlabeled CH2O. The major route of nucleic acid labeling at all concentrations and in all tissues was metabolic incorporation; protein labeling in the respiratory mucosa was mainly due to covalent binding at the higher CH2O concentrations. Incorporation of [14C]CH2O into DNA in the respiratory mucosa was maximal at 6 ppm but decreased at higher concentrations, whereas labeling of DNA in the olfactory mucosa and bone marrow increased monotonically with concentration. Evidence for covalent binding of CH2O to respiratory mucosal DNA was obtained at CH2O concentrations equal to or greater than 2 ppm. The concentration of CH2O covalently bound to DNA at 6 ppm was 10.5-fold higher than at 2 ppm, indicating significant nonlinearity of DNA binding with respect to the inhaled formaldehyde concentration under these conditions. Covalent binding to proteins increased in an essentially linear manner with increases in the airborne concentration. No evidence was obtained for the formation of covalent adducts with macromolecules in the olfactory mucosa or bone marrow. The nonlinear increase in covalent binding to respiratory mucosal DNA with increasing CH2O concentrations may be explained either by a decrease in the efficiency of defense mechanisms or by an increase in the availability of reaction sites on the DNA resulting from increased cell turnover.