Genetically unstable DNA sequences of 16.1 kb in length were isolated from the white locus of the W(mky) strain of Drosophila simulans. This insertional DNA has some unique characteristics as a transposon. It is found in high numbers in this strain and its revertant strains W(psm1) and W(cho), but not elsewhere, and the sequence is a tandem triplication of a basic repeating unit. In order to determine the structure of the insert as whole and the functional unit as a transposon, we analyzed nine clones isolated from genomic libraries of W(psm1) and W(cho). The repeating unit of the 16.1 kb insertion was the retrotransposon ninja and the DNA sequence of the entire element was determined. The ninja transposon is 6644 bp in length, with a 316 bp long terminal repeat (LTR) on each end. It contains two openreading frames (ORFs), and the pol region is divided between the two ORFs in contrast the organization of other retrotransposons. An alignment analysis of the reverse transcriptase sequences suggested that the ninja element is the first Drosophila retrotransposon belonging to the Pao subgroup.