The frequency and distribution of somatic hypermutation in immunoglobulin genes and the effect of amino acid substitution on the structure/function of antibodies were studied using hybridomas that secrete anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) monoclonal antibodies bearing lambda 1 chains. A high frequency of mutation was observed in V-J exons and J-C introns of rearranged and active lambda 1 chains but not in the 5'-non-coding regions of these chains. Since a similar distribution was observed in inactive lambda 2 chain genes, 5'-non-coding regions containing a promoter were considered to be protected from mutation in view of their apparent importance. Using transgenic mice carrying chloramphenicol acetyl transferase transgenes driven by the VH promoter and heavy-chain intron enhancer, it was also revealed that these cis-acting elements are important in the induction of somatic hypermutation and are capable of inducing mutation even in non-immunoglobulin genes. Affinity of anti-NP Abs to NP increased with time after immunization to approximately 8,000-fold (affinity maturation); however, fine specificity, such as heteroclicity, remained unchanged. Memory B cells, which are responsible for affinity maturation, were analyzed in terms of the mutation from Trp to Leu at position 33, a change known to raise affinity about 10-fold and considered to be a memory B-cell marker. These cells were found predominantly in the early stage (2-3-week) hybridomas but rarely in late stage (> 12-week) ones, suggesting that a dynamic change in the memory B-cell population occurs during the immunization process.