Current debate regarding the potential for significant reductions in human health risk associated with smokeless tobacco (ST) use compared to cigarette smoking suggests further examination of the relative cellular/molecular effects of non-combustible and combustible tobacco products is warranted. Available in vitro data regarding the potential toxicological effects of ST Jacks consensus, which prompted an investigation into the relative cytotoxic potential of ST and whether ST elicits differential cellular/molecular responses compared to combustible tobacco preparations. Cigarette smoke condensate (CSC) and whole smoke conditioned medium (WS-CM) were prepared from 3R4F cigarettes, while 10% ST extract in artificial saliva with enzymes (ST/CAS) was prepared from 2S3 moist tobacco. Two different human cell lines, HL60 and THPI, and freshly isolated human peripheral blond mononuclear cells (PBMCs) were used to examine the relative cytotoxic effects of the tobacco preparations, as well as nicotine in short-terra cell culture assays. Corresponding EC50 values, normalized for nicotine content, suggest that combustible tobacco preparations induced markedly higher cytotoxicity, as follows: WS-CM≥CSC>ST> nicotine. WS -CM and CSC similarly induced time-dependent cytotoxicity in PBMCs. While all three tobacco preparations induced some level of DNA damage, IL-8 secretion and oxidative stress, the combustible tobacco preparations were significantly more potent than ST/CAS. Moreover, tobacco preparations appeared to elicit differential responses in dendritic cell maturation assays. These findings suggest that the relative cytotoxic and other cell biological effects of tobacco preparations are dose-dependent, but that ST-induced cytotoxic effects, inflammatory responses, oxidative stress and DNA damage responses are evident only at substantially higher doses in this study.