The objective of this study was to determine the effect of smoking conditions on the in vitro toxicological activity of mainstream smoke. The 2R4F reference cigarette was machine-smoked by International Organization for Standardization(ISO) and Canadian Intense(CI) conditions. Smoke was analysed for chemical composition and in vitro toxicity. The cytotoxic potencies of both the total particulate matter(TPM), which were collected in Cambridge filter pad, and gas/vapor phase(GVP), which was bubbled through in phosphate-buffer saline in a gas-washing bottle, were assessed neutral red up take assay with chinese hamster ovary(CHO) cells. The assessment for genotoxicity of TPMs generated under ISO and CI conditions was determined using Salmonella mutagenicity assay and in vitro micronucleus assay. When calculated on an equal TPM basis, in vitro toxicity of TPM obtained under CI condition was decreased compared to TPM generated under ISO condition. The results of chemical composition analyses revealed that the lower toxicological activity under CI condition than that of ISO condition could be explained by the decreased in the contents of phenols, N-nitrosoamines and aromatic amines of TPM on an equal TPM basis