Further studies have been conducted in an attempt to define the mechanism of the intense accumulation of radioactivity in bronchial epithelium following the administration of nicotine-14C to mice. Male and female pigmented (C57B1/6J) and nonpigmented (A/HeJ) mice were studied by whole-body autoradiography following administration of either nicotine-14C (methyl- or 2'-labeled) or nicotine-1'-N-oxide-14C. When each of these compounds was administered at the same specific activity the radioactivity retained in the bronchial epithelium was much greater for the methyl-labeled nicotine-14C than for the ring-labeled. Following administration of the N-oxide very little radioactivity was retained at this site and this was seen only at a few time intervals. Pretreatment with NaHCO3, NH4Cl, SKF 525A, piperonyl butoxide, progesterone, cysteamine, phenobarbital or metyrapone prior to the administration of [methyl-14C]nicotine was studied. Metyrapone totally prevented the accumulation of radioactivity in the bronchial epithelium and progesterone reduced this accumulation; the other substances used for pretreatment had no effect on the uptake of radioactivity. These results are interpreted to indicate that the accumulation of nicotine in bronchial epithelium is not accounted for by transcellular pH gradients but is due to a high affinity of nicotine for a cytochrome P-450 in this tissue which demethylates the nicotine. The relationship and significance of metabolism of nicotine in this tissue to metabolism at other sites in the body are discussed.