In this study, we characterized the immunoglobulin light (IgL) chain gene and examined its expression in the fugu (Takifugu rubripes). The cDNA fragment that partially encodes the IgL chain was isolated by RACE and used as a probe for screening for IgL in a fugu splenic cDNA library. The IgL cDNA sequence that we found consisted of a variable (V(L)) and a constant (C(L)) segment. Its structural features were similar to the IgL isotype commonly found in teleosts. Genomic sequence analysis revealed that the IgL gene was organized as two V(L) gene segments (designed V(L1) and V(L2)) followed by single joining (J(L)) and C(L) segment. In addition, an unusual duplicate V(L1) gene segment was found downstream of the C(L) segment. The transcriptional orientation of the V(L) exons was found to be opposite to that of the J(L) and C(L) segments. Genomic blot hybridizations with V(L) and C(L) probes gave multibands, supporting the contention that the teleost IgL forms a multicluster. Both genomic and cDNA sequences analyses showed that all of the constant segments found in the fugu are identical, suggesting that no other isotypes could be found in this species. Comparison of the deduced amino acid sequence of the fugu C(L) domain with those of other species showed a high degree of identity (from 40 to 77%). IgL mRNAs were found to be expressed primarily in the lymphoid tissues. In situ hybridization revealed the presence of IgL-positive cells widely distributed throughout the spleen, head kidney, kidney, and thymus. These results support the contention that the lymphoid tissues are the major sites of antibody production in fish. Since IgL mRNA was also expressed in the skin and gill that are exposed to external antigens, it is likely that mucosal Ig plays an important role in immune protection.