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A system for the simultaneous exposure of monolayer cell cultures to mainstream (MS) and sidestream (SS) smoke from the same cigarette was utilized to study the effects of smoke generation and manipulation variables on the cytotoxicity of smoke to monolayer cultures of mouse fibroblast-like L-929 cells. The cytotoxicity of MS smoke was decreased with increasing smoke age (up to 8.7 s), smoke dilution, and the quantity of activated charcoal in filters. Acetate filters had little effect on cell mortality, and the age-of-smoke effect was not evident for MS smoke generated with a low puff volume and rapid dilution. The cytotoxicity of SS smoke also decreased rapidly with increasing smoke age and dilution. The results indicate that the gas phase of smoke may be of major importance in generating the observed toxic effects. These results may be of potential future significance in defining the requirements for a less toxic cigarette, in considering the hazards of SS smoke, and in evaluating in vivo inhalation studies.