This series of papers provides a description of the toxicological evaluation of an electrically heated cigarette (EHC). With this novel cigarette design the tobacco is heated by a series of electric heating elements, which allows for greater control of the available heat and results in lower temperatures and less combustion compared with conventional lit-end cigarettes. This design was subjected to testing, including an evaluation of smoke chemistry, in vitro bacterial genotoxicity, in vitro mammalian cell cytotoxicity and a 90-day smoke inhalation study in rats. A conventional lit-end cigarette, the University of Kentucky Reference Cigarette 1R4F, was used as a point of comparison in these experiments. When adjusted for the yield of total particulate matter, the EHC delivered 50% lower amounts of about two-thirds of the 69 smoke constituents measured. Mutagenic activity (Salmonella reverse mutation assay) of the particulate phase material in the presence of metabolic activation was ca. 90% lower, with a slight reduction of activity in the absence of metabolic activation. Cytotoxic activity (neutral red assay) of the particulate phase material was ca. 40% lower, with about equal activity of the gas/vapor-phase material. Equal activity was noted between cigarette types in a whole smoke rat inhalation assay. The results from this series of tests demonstrate that the EHC produces a much different smoke–with an at least partially reduced yield of smoke constituents and biological activity–from that of a standard reference cigarette.