The presence of multiple VHDJH joinings in upwards of 30% of acute lymphoblastic leukemias (ALL) suggests a relative instability of the rearranged immunoglobulin heavy chain (IgH) gene, but the mechanisms involved are not completely understood. An investigation of the structure of the VHDJH joinings using complementarity determining region (CDR)3 polymerase chain reaction (PCR) in 12 leukemias at both diagnosis and relapse indicates that this instability may increase as a function of time. In only one of seven cases in which relapse occurred within 3 years from diagnosis was a new VHDJH joining identified and this coexisted with the original diagnostic joining. Most strikingly, new VHDJH joinings were identified in four of five cases in which relapse occurred more than 5 years from diagnosis. In this latter population, the instability of the joinings was generated from VH----VH gene replacement events in two cases, since the new joinings retained the original DJH sequences and partial N region homology at the VHD junction, and probably in a third case from a VH gene rearrangement to a common DJH precursor. Furthermore, in five of 23 (21.7%) additional cases studied at diagnosis, subclones were identified that had similar modifications of the VH-N region. These data indicate that VH gene replacement events and VH gene rearrangements to a common DJH joining contribute to the instability of the VHDJH joining in ALL. This phenomenon should be taken into consideration in those methodologies that exploit IgH rearrangements for detection of minimal residual disease.