The water soluble fraction of the gas/vapor phase (GVP) of 2R4F cigarette mainstream smoke has been shown to be mutagenic in salmonella typhimurium strain TA100 without S9 metabolic activation in the microsuspension version of the salmonella reverse mutation assay. To estimate the contribution of single smoke constituents to the mutagenic activity of GVP from the reference cigarette 2R4F, GVP was screened for smoke constituents by a targeted GC/MS screening method and an HPLC method (formaldehyde only). 65 single smoke constituents, from 13 chemical classes were identified, with the aldehydes having the highest yields. The ten constituents with the highest yields, i.e., acetaldehyde, acetone, 2,3-butanedione, 2-butanone, acetonitrile, acrolein, propionaldehyde, methyl vinyl ketone, crotonaldehyde, and toluene, as well as five other constituents which are reported to be mutagenic, i.e., methacrolein, formaldehyde, benzene, pyridine, and 2-furaldehyde, were assayed as pure substances for their mutagenic activity. Results showed that the mutagenic activity of GVP from the 2R4F could be attributed mainly to six smoke constituents: acrolein, crotonaldehyde, formaldehyde, methyl vinyl ketone, methacrolein, and 2,3-butanedione. Of these six constituents, acrolein, crotonaldehyde, and formaldehyde account for approximately 60% of the mutagenicity.