Analysis of 10 cDNA encoding lambda L chains of horse Ig indicated that this species may employ a relatively small number of variable region (V lambda) genes in the splenic B cell population. The V lambda sequences of all of the cDNA analyzed were closely related (> 88% identity at the nucleotide level) and were characterized by a deletion of the amino acid residue at position 3 compared with V lambda sequences so far described in other species. The 10 V lambda sequences could be grouped into three groups, V lambda 1 to V lambda 3, on the basis of a number of linked substitutions. Sequences within the groups showed the greatest divergence in the third cdr regions and at the V-J junctions. The junctional variation included amino acid substitutions on both sides of the V-J junction as well as the insertion or deletion of two to four amino acid residues. Four C lambda genes were identified in genomic blots of horse DNA, and three of these were found expressed in splenic cDNA. The fourth C lambda gene may represent a pseudogene, inasmuch as the associated J region possessed a defective heptamer joining sequence. Six of the nine possible V lambda-C lambda combinations were found in the cDNA analyzed, suggesting that genes belonging to groups V lambda 1 through V lambda 3 may rearrange to any one of three J lambda-C lambda genes. One V lambda germline gene was characterized and found to represent a distinct V lambda group (V lambda 4), not represented in the cDNA sequences analyzed. The number of germline V lambda genes was estimated to be 20 to 30, based on analysis of restriction fragments hybridizing with V lambda probes. On the basis of these data, we propose that the V lambda repertoire in horse may consist of relatively limited number of genes, of which only a few may be used at high frequency in the splenic B cell population. The results indicate that predominance of lambda-chains in horse Ig may not simply be due to the presence of a large germline V lambda gene repertoire.