Tol2 is an active DNA-based transposable element identified in the medaka fish, Oryzias latipes. Originating from a vertebrate and belonging to the hAT ( hobo/ Activator/ Tam3) transposable element family, featuring a wide distribution among organisms, Tol2 would be expected to be active if introduced into mammals. We, therefore, examined if excision, one part of the transposition reaction, can occur in human and mouse culture cells. A Tol2 clone was introduced into cells and, after incubation, recovered. PCR and sequencing analysis provided evidence for precise and near precise excision in these cells. Tol2 can thus be expected to serve as a material for developing a gene transfer vector and other genetic tools applicable to mammals. It was also suggested that an intact Tol2 element could retain autonomy as a transposable element in mammalian cells.