The recombination-stimulating sequence, HOT1, corresponds to the promoter of transcription by yeast RNA polymerase I. The effect of HOT1 on mitotic interchromosomal recombination was examined in diploid strains carrying a heterozygous URA3 gene on chromosome III. The frequency of Ura- recombinants was increased 20-fold when HOT1 was inserted into the chromosome III copy marked with URA3, at a location 48 kbp centromere-proximal to URA3. Ura- recombinants were increased only 2-fold when HOT1 and URA3 were on opposite homologues. These results suggest that most HOT1-promoted Ura- recombinants result from gene conversion and that sequences on the HOT1-containing chromosome are preferentially converted. Characterization of Ura- recombinants isolated from strains carrying multiple markers on chromosome III indicates that HOT1-promoted gene conversion tracts are unusually long (often greater than 75 kbp) and almost always continuous. Furthermore, conversion tracts frequently extend to both sides of HOT1. We suggest that HOT1 promotes the formation of a double-strand break which is often followed by exonucleolytic digestion. Repair of the broken chromosome could then result from gap repair or from replicative repair primed only by the centromere-containing chromosomal fragment.