The FHIT gene spans approximately 1 Mb of DNA at chromosome band 3p14.2, which includes the familial renal cell carcinoma chromosome translocation breakpoint (between FHIT exons 3 and 4), the most frequently expressed human constitutive chromosomal fragile site (FRA3B, telomeric to the t(3;8) translocation), and numerous homozygous deletions in various human cancers, frequently involving FHIT exon 5. The FRA3B has previously been shown to represent more than one specific site, and some specific representatives of FRA3B breaks have been shown to fall in two regions, which we know to be in FHIT introns 4 and intron 5. Because breakage and integration of exogenous DNA in this chromosome region is frequent in aphidicolin-treated somatic cell hybrids, cancer cells, and, presumably, aphidicolin-treated normal lymphocytes that exhibit gaps or breaks, we determined by one- and two color fluorescence in situ hybridization, using cosmids covering specific regions of the FHIT gene, that most of the aphidicolin-induced gaps at FRA3B fall within the FHIT gene, with the highest frequency of gaps falling in intron 5 of the FHIT gene, less than 30 kb telomeric to FHIT exon 5. Gaps also occur in intron 4, where a human papillomavirus 16 integration site has been localized, and in intron 3, where the t(3;8) break point is located. These results suggest that the cancer-specific deletions, which frequently involve introns 4 and 5, originated through breaks in fragile sites.