Little cigars (also known as cigarillos) have been increasing in popularity with respect to cigarettes; but relatively little is known about the toxicology of the mainstream smoke (MSS) from little cigars. Therefore, the objective of this study was to compare the toxicological properties of the MSS (Health Canada Intensive smoking conditions) from a range of little cigar products with the toxicological properties of MSS of other smoking products such as cigarettes. Three in vitro assays were used to evaluate the toxicities of the MSS total particulate matter (TPM): 1) mutagenicity using Ames assay with strains TA98 and TA100 with S9 metabolic activation (+S9); 2) cytotoxicity using the Neutral Red assay with CHO cells; and 3) genotoxicity using the micronucleus assay with CHO cells and short-term exposures (3-hour ±S9). The Ames assay (TA100+S9) and the Neutral Red assay were also applied to the gas/vapour phase (GVP) of the MSS that passed through the Cambridge pad. On a per-milligram nicotine basis, which is one way of comparing toxicities of different types of tobacco products, TPM from the little cigars was more mutagenic with TA98+S9 and TA100+S9 than was the TPM from cigarettes (70.6±11.5K and 26.5±7K versus 34.4±5K and 16.1±3K revertants/mg-nicotine). A similar but less pronounced trend was noted with the micronucleus results. The GVP fractions for some little cigars were more mutagenic with TA100+S9 than were the corresponding GVP fractions from other little cigars and cigarettes, which showed very little mutagenicity. The TPM and GVP smoke fractions from the little cigars were more cytotoxic than were the corresponding smoke fractions from cigarettes (TPM IC50 1.80±0.35 versus 3.40±0.56 mg nicotine/mL, GVP IC50 3.73±0.70 versus 5.16±0.65 mg nicotine equivalent/mL). Thus, our findings support our prior work on smoke mutagenicity that showed MSS from little cigars was not less toxic than MSS from cigarettes.