Cigarette smoke condensates (CSCs) are complex mixed compounds that contain both direct and indirect mutagens/carcinogens. To detect genotoxicity of CSCs in vitro, a combination of various enzymes (e.g. activation and detoxification enzymes) called S9 is usually added. However, as S9 may induce cytotoxicity in target cells, it is unclear whether the addition of S9 can impact CSC-induced toxicity. Here, differences in cytogenotoxicity between CSCs in the presence or absence of S9 were studied using three in vitro assays (neutral red uptake assay, comet assay, and TCR gene mutation test) in human peripheral lymphocytes, which were exposed to CSCs at doses of 25, 50, 75, 100 and 125μg/ml for 4h. Assay results showed that both CSCs+S9 or CSCs−S9 could induce a dose-dependent elevation of cytogenotoxic effects in human lymphocytes with some differences between the two groups. The cytogenotoxicity induced by CSCs−S9 was significantly higher than that induced by CSCs+S9 in all three assays. The comet and NRU assays revealed that a dose–response relationship of cytogenotoxicity induced by CSCs+S9 was less typical than that induced by CSCs−S9, possibly due to specific cytogenotoxic agents in CSCs and enzymes contained in the S9 mixture. Thus, the three in vitro assays used in the present study are suitable for detecting cytogenotoxic effects in human lymphocytes induced by CSCs. Furthermore, the cytogenotoxicity induced by both CSCs+S9 and CSCs−S9 should be measured simultaneously when assessing and comparing the biological activity of different CSCs.