Earlier studies distinguished two classes of catfish light (L) chain (designated F and G). The cDNA structure and genomic organization of G L chain gene clusters has also been characterized previously. In this study, full length cDNA encoding F L chain was derived using PCR strategies based on the determined amino-terminal protein sequence. The encoded V region is readily delineated into framework regions (FR) and complementarity-determining regions (CDR). Multiple sequence alignments indicate that the F V(L) is closely related to kappa gene families. The F C(L) cannot be generally classified but it is structurally distinct from the C(L) regions of G: the amino acid sequence similarity is <35%. cDNA sequences representing processed sterile F transcripts of different loci were identified. Each sequence begins within the J(L) recombination signal sequence and extends downstream through the I(L)-C(L) segments. Genomic blots hybridized with C(L) probes indicate that there are at least 50 different C(L) segments. Based upon V(L) hybridization studies, different families of V(L) segments appear to be associated with closely related F C(L) segments. In characterized genomic clones, F gene segments are arranged in closely linked clusters with single copies of V(L), J(L), and C(L) segments within each cluster. The V(L) segments are located in opposite transcriptional polarity relative to the J(L) and C(L) segments, which indicates that V(L) segments rearrange by inversion. These combined studies establish that two structurally distinct classes of L chains are present in teleost fish and that both of the L chain classes evolved within a common organizational pattern of clustered segmental genes.