The common fragile site at 3p14.2 (FRA3B) has been described as the most active fragile site in the human genome. This locus may predispose chromosome 3 to specific losses due to deletions and translocations that have been associated with several malignancies, including hereditary renal cell carcinoma. We have previously described induction of breakage around FRA3B using aphidicolin in a somatic cell hybrid whose only human component was a single intact chromosome 3. That work led to the isolation of hybrids with breakpoints in the 3p13-p21.1 region with loss of all sequences distal to their respective breakpoints. In this report we describe the further characterization of the breakpoints in many of these cell lines using newly available molecular markers. We also report the identification of YAC clones that span the breakpoints present in many of these hybrids.