Based on the analysis of a panel of variable (V) region sequences from the Australian duck-billed platypus and the Australian short beaked echidna, the monotremes were found to express a highly diversified Vk repertoire. High variability was observed both in sequence and in length of all three CDR regions. However, all monotreme sequences were found to form a separate branch on a distance tree, and the monotremes appear to express only two Vk gene families. The appearance of all Vk gene segments in one branch on the distance tree gives further support for the notion that deletions of entire V region clans or families, followed by successive rounds of gene duplications may be a relatively common phenomenon during vertebrate evolution. Four different constant region sequences were also identified and a preferential use of certain J segments to each constant region was observed. A more detailed picture of the locus was obtained by analysis of genomic DNA by Southern blot and PCR. The organization of the lambda locus involves multiple V and several constant region genes with one or several joining segments positioned upstream of each constant region, similar to the organization in mouse and man. An mRNA frequency analysis shows that the k light chain accounts for more than 90% of the light chain transcripts in the spleen. The abundance and the high variability indicate that light chain diversity at the k locus contributes significantly to the antigen-binding repertoire in monotremes. A high k to j light chain ratio also indicates that variability in the CDR regions is more important for the repertoire size than the total number of V gene families.