Chlorine gas is a respiratory irritant in both animals and humans that produces concentration-dependent responses ranging from minor irritation to death. Female and male B6C3F1 mice and F344 rats were exposed to chlorine gas for up to 2 years to determine chronic toxicity and carcinogenicity. Groups of approximately 70 each of female and male mice and rats were exposed to 0, 0.4, 1.0, or 2.5 ppm chlorine gas for 6 hr/day, 5 days/week (mice and male rats), or 3 alternate days/week (female rats) for 2 years, with an interim necropsy of rats at 12 months (10 rats/sex/concentration group). A complete necropsy was performed on all animals. Histological examination was performed on all organs from high-concentration and control animals and selected target organs from mid- and low-concentration groups. Exposure-dependent lesions were confined to the nasal passages in all sex and species groups. Chlorine-induced lesions, which were most severe in the anterior nasal cavity, included respiratory and olfactory epithelial degeneration, septal fenestration, mucosal inflammation, respiratory epithelial hyperplasia, squamous metaplasia and goblet cell hypertrophy and hyperplasia, and secretory metaplasia of the transitional epithelium of the lateral meatus. Intracellular accumulation of eosinophilic proteinaceous material was also a prominent response involving the respiratory, transitional, and olfactory epithelia, and in some cases the squamous epithelium of the nasal vestibule. Many of these nasal lesions exhibited an increase in incidence and/or severity that was related to chlorine exposure concentration and were statistically significantly increased at all chlorine concentrations studied. Male mice and female rats appeared more sensitive to chlorine than female mice and male rats, respectively. The reasons for the sex differences within a species were not determined. Interspecies differences in regional dosimetry and site-specific tissue susceptibility to chlorine exposure should be taken into account when using these data for accurate assessment of potential human health risks. The incidence of neoplasia was not increased by exposure, indicating that inhaled chlorine in rats and mice is an upper respiratory tract toxicant but not a carcinogen.