Chromosomal or allelic losses at 3p14 are common in a variety of human tumors, including those of the lung, breast, kidney, and head and neck. This suggests the existence of a tumor suppressor gene in this band. A promising candidate is the recently cloned FHIT gene, which spans the common fragile site, FRA3B, at 3p14.2. We previously identified a region of fragility at 3p14.2 (FRA3B) of > 85 kb by cloning DNA flanking pSV2neo integrations and constructed a partial genomic contig of the region. Using probes from the contig, we tested for deletions within this region in DNA from 105 human tumor cell lines, predominantly derived from lung cancers. We identified one gastric and four lung cancer cell lines with homozygous interstitial deletions involving the FRA3B region. The deletion in one lung cancer cell line lies entirely within our contig and is < 65 kb. We have identified, cloned, and sequenced this breakpoint junction. We have also shown that our probes lie within intron S of the FHIT gene and, furthermore, that exon 5 is located approximately 1 kb from one of our probes and, thus, lies within the region of fragility. Two lines with entirely intronic deletions yield FHIT transcripts of normal size. In one of these, this was the sole transcript identified. In the other line, an FHIT transcript completely normal in sequence was accompanied by two larger abnormal transcripts. These results leave open the possibility that some homozygous deletions within the FHIT gene are without phenotypic effect and result from genetic instability of this region. However, taken together, our results provide evidence that breakage and rearrangement within the FRA3B fragile site sequences result in alterations of FHIT and are likely to be involved in carcinogenesis.