CD19+ B lineage acute lymphoblastic leukemias (ALLs) with unrearranged Ig and TCR genes are designated germline B lineage ALLs. We used CDR3 PCR to determine whether pediatric germline B lineage ALLs contain minor subclones with rearranged Ig H V genes. In six of seven cases there were no PCR detectable CDR3 rearrangements. One case with a smear pattern on CDR3 PCR contained multiple unique CDR3 sequences at frequencies of 1-2 per 2,600, suggesting that polyclonal B cells were present at low frequency. To verify that the germline patterns were from leukemic cells and evaluate in vivo subclone differentiation, a germline B lineage ALL with the t(4;11) translocation was propagated in severe combined immunodeficient SCID) mice. The Ig and TCR genes in the leukemic cells recovered from mouse tissues were germline by Southern blot analysis except for single rearrangements that suggested subclone evolution at the Ig H and lambda loci in addition to the germline population. No CDR3 sequences were detected, indicating that the observed Ig H gene rearrangement most likely was a DJ joining. This study suggests that the transformed cell in germline B lineage ALL represents an early pro-B cell with limited tendency to further differentiate.