Sequence analysis of the immunoglobulin heavy chain complementarity determining region (CDR)-III of B-lineage cells at various stages has provided important insights concerning B cell maturation and selection. Knowledge of human CDR-III sequences has been relatively limited compared with that of the murine system. We analyzed the CDR-III sequences of B cell precursor acute lymphoblastic leukemia (pre-B ALL) cells in 23 newly diagnosed and 10 relapsed patients, in order to elucidate the organization of CDR-III in B cell precursors. We found a very low frequency of somatic mutations in D and JH regions, preferential use of DLR, DXP, DHQ52, and DN elements, and of 3' side JH segments, and no predominant usage of D coding frames. Unusual joinings such as VH-D-D-JH and VH-JH were observed in three, and one sequences, respectively. We compared the CDR-III sequences derived from 10 patients between diagnosis and relapse. Two of them had three spots of mutated nucleotides at relapse, all of which were found in the N region near the D segments. Our data showed the possibility of somatic mutation at relapse, in addition to developmentally regulated rearrangement of the immunoglobulin gene at the stage of B cell precursors.