The distributions of nicotine-14C (2''- or methyl-labeled), cotinine-14C, and nicotine-1''-N-oxide-14C were studied by whole-body autoradiography in mice and in perfused rabbit lung. The compounds were administered either i.v., s.c., i.p. or by inhalation. Blank sections were also incubated with nicotine-14C in vitro. The distributions were compared in a black (C57BL/6J), brown (C57L/J) and albino (A/HeJ) strain and in CD-1 germ-free mice. After administration of nicotine-14C in vivo, radioactivity was localized in all strains in bronchi, nasal mucosa, salivary gland. Harder''s gland, liver, kidney, stomach, spleen, pancreas, intestine, bone, gallbladder and adrenal medulla. In the pigmented strains, it was also localized in melanin in the eye, brain and hair. Radioactivity did not accumulate in the bronchi of late-term fetuses or the 1 day old newborn but was present in the 2 day old and older. Cotinine-14C and nicotine-1''-N-oxide-14C, after i.v. administration, did not localize in bronchi or nasal mucosa at short time intervals after injection. Frozen sections incubated in vitro did not accumulate either nicotine-14C in the bronchi; whereas the affinity for melanin was unchanged. Incubation of fresh, nonfrozen mouse lung and perfusion of rabbit lung in vitro with nicotine-14C produced the same localization of radioactivity in bronchi as that seen after in vivo administration. Preheating the frozen sections or lungs in a microwave oven before incubation did not change the localization of radioactivity in the bronchi or other tissues. The localization of radioactivity in bronchi may be due to metabolism, active transport or binding of nicotine; this mechanism was destroyed by freezing the tissue.