Vanillin is a flavoring agent used in cigarettes. Previous toxicological examinations of the effects on the addition of vanillin to tobacco used mixtures with several other flavoring agents. In the present work, toxicological comparisons were made of experimental cigarettes containing no added vanillin against otherwise similar cigarettes with three different amounts of vanillin added to the tobacco. The main toxicological comparison was a subchronic inhalation study with mainstream smoke in Sprague-Dawley rats (exposures of 150 mg/m3 of total particulate matter, 6 h exposure per day, for 90 consecutive days). Vanillin concentrations in the tobacco of the 4 cigarette types at the end of the study were 0, 67, 1233, and 3109 ppm. Additional studies with mainstream smoke were Salmonella mutagenicity (5 bacterial strains, both with and without metabolic activation, particulate phase only), cytotoxicity of both particulate and gas/vapor phases (using the neutral red uptake assay), and analytical chemistry (49 analytes, including 5 metals). Similar responses were seen across the four cigarette types, and the responses were similar to those previously described in the scientific literature. At the same smoke concentration, the inhalation exposures produced effectively the same responses, in each of the four groups. Most of the changes produced in the 90 days of exposure were resolved in a 42-day postinhalation period. The addition of vanillin to tobacco at inclusion rates up to 3109 ppm did not influence a broad range of toxicological endpoints.