We have characterized the genomic organization of the TyS retrotransposons among diverse strains of Saccharomyces cerevisiae and the related species Saccharomyces paradoxus. The S. cerevisiae strain S288C (or its derivatives) carries eight Ty5 insertions. Six of these are located near the telomeres, and five are found within 500 bp of autonomously replicating sequences present in the type X subtelomeric repeat. The remaining two S. cerevisiae elements are adjacent to the silent mating locus HMR and are located within 500 bp of the origin of replication present in the transcriptional silencer HMR-E. Although the S. cerevisiae TyS elements no longer appear capable of transposition, some strains of S. paradoxus have numerous Ty5 insertions, suggesting that transposition is occurring in this species. Most of these elements are adjacent to type X telomeric repeats, and regions flanking four of five characterized S. paradoxus insertions carry autonomously replicating sequences. The genomic organization of the Ty5 elements is in marked contrast to the other S. cerevisiae retrotransposon families (Tyl-4), which are typically located within 500 bp of tRNA genes. For Ty3, this association reflects an interaction between Ty3 and the RNA polymerase III transcription complex, which appears to direct integration [Chalker, D. L. & Sandmeyer, S. B. (1992) Genes Dev. 6, 117-128]. By analogy to Ty3, we predict that Ty5 target choice is specified by interactions with factors present at both the telomeres and HMR that are involved in DNA replication, transcriptional silencing, or the maintenance of the unique chromatin structure at these sites.Retrotransposons are mobile genetic elements that replicate by reverse transcription of an RNA intermediate. The final step in the retrotransposon life cycle is the integration of an element into a new site in the host genome. Integration may be deleterious to the host cell because it can cause mutations or lead to chromosome rearrangements. Mechanisms that minimize this damage preserve the host's genetic integrity and also allow retrotransposons to replicate and maintain viable populations. In the yeast, Saccharomyces cerevisiae mechanisms have evolved that limit the negative consequences of integration by directing transposition to specific sites in the genome.Target preference of the S. cerevisiae retrotransposons has been made strikingly evident by the recent genome sequencing efforts. Insertions of the four best-characterized retrotransposon families (Tyl-4) are found within the nucleotide sequences of chromosomes III and XI (chr III and chr XI, respectively)