The Tol2 element is found in the genome of the medaka fish, Oryzias latipes, and contains DNA sequences similar to those of transposons of the hAT family. Previously, we have developed a transient embryonic excision assay in zebrafish, in which zebrafish embryos were injected with a plasmid DNA harboring the Tol2 element, and have shown that the Tol2 element is excisable from the injected plasmid DNA (Kawakami, K., Koga, A., Hori, H., Shima, A., 1998. Excision of the Tol2 transposable element of the medaka fish, Oryzias latipes, in zebrafish, Danio rerio. Gene 225, 17-22). Although the Tol2 element is thought to be autonomous, an active transposase encoded by the Tol2 element has not been identified. Here we report the identification and analysis of mRNA transcribed from the Tol2 element in zebrafish embryos. The Tol2 transcript has the capacity to encode a protein of 649 amino acids, whose amino acid sequence is similar to those of transposases of the hAT family. To determine whether the transcript encodes an active enzyme, we developed a novel transient embryonic excision assay in which zebrafish fertilized eggs were co-injected with RNA transcribed in vitro using the Tol2 cDNA as a template and a plasmid DNA harboring a nonautonomous Tol2 element, which has a deletion in the transposase coding region. The nonautonomous Tol2 element could be efficiently excised in the zebrafish only when co-injected with the Tol2 RNA. This result indicates that the Tol2 transcript encodes an active enzyme, a probable transposase, that can catalyze the excision reaction in trans. Further, by the co-injection analysis, we found that the Tol2 sequence lacking the first intron sequence of the transposase gene could not be excised, suggesting that it may contain essential cis-elements.