We determined the immunoglobulin (Ig) V(H) subgroup expressed by the leukemia cells of 108 patients with B cell chronic lymphocytic leukemia (CLL). Surprisingly, we found that six samples (5%) each expressed Ig of more than one V(H) subgroup. Southern blot analysis demonstrated that these samples each had rearrangements involving both Ig heavy chain alleles. Nucleic acid sequence analyses of the Ig cDNA revealed each to express two functional Ig V(H) genes: V(H)3-33 and V(H)4-39; V(H)3-7 and V(H)4-39; V(H)3-23 and V(H)4-61; V(H)2-70 and V(H)3-30.3; or V(H)3-30 and V(H)4-b (DP67). One sample expressed three Ig V(H) genes: V(H)2-70, V(H)3-7, and V(H)4-59. Despite having more than one Ig heavy chain transcript, each sample was found to express only one functional Ig light chain. From the primary sequence, we deduced that the Ig of some of these CLL samples should react with Lc1, a monoclonal antibody (mAb) reactive with a supratypic cross-reactive idiotype present on Ig encoded by a subgroup of Ig V(H)4 genes (namely, V(H)4-39, V(H)4-b [DP-67], V(H)4-59, or V(H)4-61), and B6, an mAb that reacts with Ig encoded by certain Ig V(H)3 genes (namely, V(H)3-23, V(H)3-30, or V(H)3-30.3), and/or modified staphylococcal protein A (SpA), a 45-kilodalton bacterial "superantigen" that reacts with most Ig of the V(H)3 subgroup. Flow cytometric analyses revealed that such samples did in fact react with Lc1 and B6 and/or SpA, but not with control mAbs of irrelevant specificity. This study demonstrates that a subset of CLL patients have leukemic B cells that express more than one functional Ig heavy chain.