Despite several lines of evidence suggesting that common chromosomal fragile sites are biologically important as hot spots for recombination, their structure remains unknown. We showed previously that the plasmid pSV2neo preferentially integrates into bands containing fragile sites in cells transfected under conditions of fragile site induction. Here we report the isolation and characterization of the DNA sequences from two such independent integrations into 3p14.2, a common fragile site (FRA3B). These FRA3B region sequences were shown to lie within a 1330-kb YAC, 850A6, approximately 350 kb telomeric of the breakpoint of t(3;8), a constitutional rearrangement. The two integration sites are 10 kb apart, but each integration is associated with a deletion. We have constructed a partial genomic contig of the integration sites and deleted regions spanning approximately 85 kb. Analysis of the DNA sequences immediately surrounding the plasmid integrations revealed no known coding sequences or repeat structures resembling the (CGG)n motif characteristic of the rare fragile sites. In addition, by Southern blotting analysis, none of the phage clones isolated from the FRA3B region were found to contain CGG repeats. Fluorescence in situ hybridization analysis of genomic clones from this contig to metaphase cells induced to express breaks demonstrated hybridization adjoining the chromosome breaks, and occasionally the hybridization signal spanned the break. The results imply that breakage occurs at variable positions within a large region (at least on the order of 85 kb). Together, these data suggest that the structure of FRA3B differs from that of rare fragile sites.