The mutagenic activity of the root canal sealer AH26 was tested in the V79/hprt mammalian cell mutation assay. AH26 was mixed and eluted immediately after mixing or after setting times of 24 h or 7 days. Different amounts of dimethyl sulfoxide or physiological saline eluates were then tested for mutagenicity. Eluates of mixed AH26 were toxic and mutagenic, and both effects strongly depended on the setting time. The number of mutants after exposure to eluates of unset AH26 was enhanced approximately 7- to 10-fold. However, the mutagenic activity of the mixed material was clearly reduced after a setting time of 1 wk. Physiological saline eluates of the mixed AH26 were toxic at higher doses, but were not found to be mutagenic. Dimethyl sulfoxide eluates of the liquid component of AH26 elicited mutagenic effects similar to the freshly mixed material; eluates made in physiological saline were barely mutagenic at a 10-fold higher concentration. In accordance with recommendations of international guidelines, the V79/hprt mammalian cell mutation assay will be routinely used for the evaluation of the mutagenicity of dental materials in further investigations.