We have identified a new superfamily of vertebrate short interspersed repetitive elements (SINEs), designated V-SINEs, that are widespread in fishes and frogs. Each V-SINE includes a central conserved domain preceded by a 5Ј-end tRNA-related region and followed by a potentially recombinogenic (TG) n tract, with a 3Ј tail derived from the 3Ј untranslated region (UTR) of the corresponding partner long interspersed repetitive element (LINE) that encodes a functional reverse transcriptase. The central domain is strongly conserved and is even found in SINEs in the lamprey genome, suggesting that V-SINEs might be ∼550 Myr old or older in view of the timing of divergence of the lamprey lineage from the bony fish lineage. The central conserved domain might have been subject to some form of positive selection. Although the contemporary 3Ј tails of V-SINEs differ from one another, it is possible that the original 3Ј tail might have been replaced, via recombination, by the 3Ј tails of more active partner LINEs, thereby retaining retropositional activity and the ability to survive for long periods on the evolutionary time scale. It seems plausible that V-SINEs may have some function(s) that have been maintained by the coevolution of SINEs and LINEs during the evolution of vertebrates.[The sequences reported in this paper have been deposited in the DDBJ/GenBank database under accession nos. AB072981-AB073004. Supplemental figures are available online at http://www.genome.org.]Retroposons are genetic elements that have moved within and among genomes via an RNA intermediate (Weiner et al. 1986). They include SINEs, LINEs, LTR-retrotransposons, and retroviruses. L1 and Alu are representative of the LINEs and SINEs, respectively, in the human genome (Weiner et al. 1986;Eickbush 1994;Smit 1996). An analysis of the entire human genome revealed that retroposons constitute up to 40% of the genome and that protein-coding sequences account for only about 1.5% (International Human Genome Sequencing Consortium 2001). The above mentioned 40% of the human genome includes several clearly discernible groups of retroposons, such as L1, LINE2, Alu, and MIR, and these easily recognizable sequences represent the results of relatively recent amplifications over the past 200 Myr (International Human Genome Sequencing Consortium 2001). Accordingly, the proportion of the genome that originated from retroposons may increase up to as much as 60% to 70%, if we could identify and include ancient retroposons, which might have been amplified in the very distant past, are now barely recognizable as such, and are buried in the genome as debris. Such a putative extensive contribution of retroposons to the construction of the contemporary human genome has not been anticipated.Typical SINEs vary from 100 to 500 bp in length. They have an internal RNA polymerase III (pol III) promoter but no open reading frames (Okada 1991;Schmid and Maraia 1992;Deininger and Batzer 1993;Okada and Ohshima 1995;Schmid 1998). Many families of SINEs have been identified in multic...