The method of in vitro cytotoxicity assay for mainstream cigarette smoke was established by using VITROCELL^® whole smoke exposure system associated with neutral red uptake assay. The cytotoxicity of mainstream cigarette smoke of 29 domestic and imported commercial available brands was evaluated by this method under Health Canada Intensive (HCI) smoking regime. The results indicated that: 1) A well dose-response relationship between cigarette smoke doses and cell viability could be obtained by the established method. 2) The stability of the method was satisfactory with the values of CV below 25 %. The established method of whole smoke exposure could be applied to evaluate the cytotoxicity of mainstream cigarette smoke.