The large interallelic distances between the three rabbit Ig V(H)a lineages, a1, a2 and a3, suggest that the persistence time of the V(H)a polymorphism could amount to 50 million years, which is much longer than that of MHC polymorphisms. Rabbit originated in the Iberian Peninsula where two subspecies coexist, one of which is confined to Southwestern Iberia (Oryctolagus cuniculus algirus). We studied the V(H) loci in the original species range to obtain a better understanding of the evolutionary history of this unusual polymorphism. Serological surveys revealed that sera from the subspecies algirus, when tested with V(H)a locus-specific alloantisera, showed either cross-reactivity ("a-positive" variants) or no reaction at all ("a-blank"). Using RT-PCR, we determined 120 sequences of rearranged V(H) genes expressed in seven algirus rabbits that were typed as either a-positive or a-blank. The data show that the V(H) genes transcribed in a-positive rabbits are closely related to the V(H)1 alleles of domestic rabbits. In contrast, a-blank rabbits were found to preferentially use V(H) genes that, although clearly related to the known V(H)a genes, define a new major allotypic lineage, designated a4. The a4 sequences have hallmark rabbit V(H)a residues together with a number of unprecedented amino acid changes in framework region 2 and 3. The net protein distances between the V(H)a4 and the V(H)a1, a2, and a3 lineages were 20, 29, and 21% respectively. We conclude that at least four distantly related lineages of the rabbit V(H)a locus exist, one of which seems to be endemic in the Iberian range.