Analysis of predominant, approximately 700,000 dalton serum antibody (Ab) of the channel catfish by SDS-PAGE revealed only one molecular mass species of heavy (H) chain (approximately 70,000 daltons), whereas a marked heterogeneity in the relative mobilities of the light (L) chains was evident. Three molecular mass L chain variants of approximately 26,000, approximately 24,000, and approximately 22,000 daltons were observed. The relationships between the L chain variants were not clear until mouse monoclonal Ab (mAb) reactive with catfish immunoglobulin (Ig) were developed. Two of these mAb, designated 3F12 and 1G7, were found to independently recognize different populations of catfish Ig. In addition, when these two mAb were used in combination they immunoprecipitated greater than 95% of specifically purified catfish anti-DNP or anti-fluorescein Ab. The L chains of catfish Ig recovered from immunoabsorbent affinity matrices conjugated with either mAb 3F12 or 1G7 were found to be clearly distinct. Monoclonal Ab 3F12 reacted with a subpopulation of catfish Ig that contained the approximately 24,000 and approximately 22,000 dalton L chain variants, whereas mAb 1G7 reacted with another subpopulation of catfish Ig that contained only the approximately 26,000 dalton L chain variant. Solid phase plate binding assays with the use of mildly reduced catfish Ig H and L chains showed that both of these mAb preferentially reacted with catfish L chains. Subsequent analysis of the two antigenically distinct L chain populations by peptide mapping procedures demonstrated that these L chains were structurally different. Furthermore, analysis of the serum from individual catfish showed the presence of both L chain types in each of 25 catfish examined. These studies strongly suggest that distinct L chain classes have evolved in fish.