Sequence diversity in the human antibody repertoire is generated in two steps: by the combinatorial assembly of V gene segments and by somatic hypermutation. Here, we have characterised these processes for the lambda (λ) light chain using a library of 7600 λ cDNA clones from peripheral blood lymphocytes. By hybridisation and sequencing we found that most λ chains are derived from the cluster of V λ segments closest to the J λ -C λ pairs and that there is considerable variation in the use of individual V λ segments (ranging from 0.02% to 27%): three of the 30 functional V λ segments encode half the expressed V λ repertoire. As a result of these biases, sequence diversity in the primary repertoire is focused at the centre of the antigen binding site. By contrast, somatic hypermutation spreads diversity to the periphery. Comparison with the human kappa (κ) light chain indicates that both κ and λ use the same strategy for searching sequence space and have almost identical patterns of diversity in the mature antibody repertoire.