To understand whether the distinct VHDJH gene utilization by natural polyreactive Abs reflects the developmentally restricted Ig VHDJH rearrangements putatively expressed by B-1 cells, we generated 11 (8 IgM, 1 IgG3, 2 IgA1), 7 (6 IgM, 1 IgG1), and 7 (2 IgM, 3 IgG1, 2 IgG3) mAb-producing lines using B-1a (surface CD5+, CD45RAlow), B-1b (surface CD5-, CD45RAlow, CD5 mRNA+), and B-2 (surface CD5-, CD45RAhigh, CD5 mRNA-) cells, respectively, sorted from adult human peripheral blood. Most B-1 a and B-1b, but no B-2, cell-derived mAbs were polyreactive; i.e., they bound different self and foreign Ags with different affinities. B-1 a and B-2 mAbs preferentially utilized VH4 (p = 0.003) and VH3 (p = 0.010) genes, respectively. All three mAb populations utilized DXP, DLR, DN DH genes, and JH6, but no mAb utilized DHQ52. There were fewer unencoded nucleotide (N) additions in the VHDJH junctions of B-1b (3.00 ± 2.52, mean ± SD) than of B-1a (12.45 ± 3.93, p = 1.23 × 10-5) or B-2 (8.29 ± 4.75, p = 0.020) mAbs. Partly due to the fewer N additions and a paucity of D-D fusions, the B-1b mAb CDR3s were significantly shorter than the B-1a mAb CDR3s (p = 0.013), which contained a nonrandom Tyr distribution (p = 0.003). Finally, all but two B-1 cell-derived mAbs were mutated, in a fashion similar to that of the Ag-selected B-2 mAbs. Thus, in the human adult, B-1 cells that make natural polyreactive Abs may not be representative of the predominantly B-1 developmental waves of colonization of the fetal and neonatal B cell repertoires, and are somatically selected.