Recently, we reported evidence for the existence of an immunoglobulin lambda light chain (lambda x) whose variable region differs from those encoded by the known V lambda gene segments V lambda 1 and V lambda 2. Expression of lambda x was detected in some hybridomas elicited by treatment of a BALB/c mouse with rabbit anti-lambda 2 antibodies coupled to bacterial lipopolysaccharide [Sanchez, P. & Cazenave, P.-A. (1987) J. Exp. Med. 166, 265-270]. We constructed a cDNA clone from one hybridoma (B6) that expresses the lambda x chain and determined the complete nucleotide sequence. The deduced amino acid sequence of V lambda x is 30-33% identical with those encoded by V lambda 1 and V lambda 2 and by V kappa gene segments. The third hypervariable region of V lambda x is four codons longer than those of the other murine variable gene segments. The expression of lambda x requires a genomic rearrangement that juxtaposes the V lambda x gene with the J lambda 2-C lambda 2 joining-constant gene pair. Rabbit anti-V lambda x antibodies detected the lambda x light chain in the normal sera of all laboratory mice tested. Lambda x expression seems to be independent of lambda 1 expression, since both SJL and SJA strains, which are defective in lambda 1 production, express normal levels of lambda x chain.