Somatic mutation of antibody variable region genes is a hallmark of secondary responses. Most often the coexpressed VH and V kappa genes of a B cell mutate at nearly equal rates. We have previously identified hybridomas from two B cell clones that exhibited > 10-fold lower frequency of mutation in their expressed V kappa 12 or V kappa 1A genes relative to their coexpressed VH genes. To gain insight into the mechanism(s) responsible for this low frequency V kappa mutation, we determined the frequency of mutation in non-coding flanking DNA from multiple members of each clone. We find a low frequency of mutation in the 5' (4/700) and 3' (1/670) non-coding regions of the expressed V kappa 1A genes consistent with the low frequency of coding region mutation. In contrast, the distribution and frequency of mutation surrounding the expressed V kappa 12.37 gene are unusual. Among the six members of this clone there are 31 mutations 3' (31/2100 bp) and no mutations 5' (0/2010 bp) of the V kappa exon. This V kappa exon has acquired mutations that are intermediate in number to its flanking regions and are significantly skewed in distribution to the 3' end. None of the 31 3' mutations are shared by two or more members of this clone, indicating that they all occurred late in clonal expansion. These results raise the possibility that some V kappa genes may lack functional cis-regulatory elements which direct V kappa coding region mutation.