Screening of a mink cDNA library with an antibody probe resulted in the isolation of clone pIGL-2 containing an Ig lambda chain coding sequence. The sequence comprised almost the entire V segment as well as J, C, and 3'-untranslated sequences. A second clone, pIGL-10, was isolated by rescreening the cDNA library with the use of pIGL-2 as a probe. pIGL-10 was found to contain a frameshift deletion of a single nucleotide in the C region. pIGL-2 and pIGL-10 were 81% homologous to each other in the FR3 of the V segment, and 95% of homology was found in their C regions. The J segments of the two clones differed in only one nucleotide position. Comparison of cloned lambda chain sequences with those of other mammals revealed that mink V lambda and C lambda genes have the highest homology with their human counterparts. The V lambda sequence of clone pIGL-2 appears to be a homologue of human subgroup III V lambda genes. Southern blot hybridization of mink DNA with the C lambda and V lambda probes derived from pIGL-2 revealed five or six hybridizing C lambda fragments and at least 11 hybridizing V lambda fragments. This suggested that the lambda genes in carnivores, like those in primates, have duplicated extensively during evolution.