Acute promyelocytic leukemia (APL) has been characterized by 15;17 chromosomal translocation, which involves the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the PML gene on chromosome 15. The breakpoints have been mapped to three cluster regions in the PML gene, and to RARA gene intron 2. We have examined the distribution of breakpoints within RARA gene intron 2. An extremely restricted region (ERR) of 50 bp within RARA gene intron 2 was identified as the duster region of breakpoints by polymerase chain reaction and sequence analysis of DNA from APL patients. To study experimentally the mechanism involved in the translocation, ERR was tested in NIH3T3 cells by in vitro transfection-recombination assay, in which target sequences were placed either downstream of the SV40 promoter or upstream of the neo gene. Cells were conferred resistance to G418 only when the promoter was fused to the neo gene by recombination of two target sequences during transfection. The molecular junctions were analysed in five clones, and all of them were shown to be confined within a 20 bp region in a 148 bp DNA fragment containing ERR. This suggests that ERR might be the illegitimate recombination hot spot in mammalian cells.