The functional affinities of antibodies of different IgG subclasses were measured in normal individuals during the primary, secondary and tertiary immune responses to keyhole limpet haemocyanin (KLH). IgG1 responses showed marked affinity maturation between Days 7 and 21 after primary and by Day 7 after secondary and subsequent immunizations, correlating with an expansion of high-affinity antibody populations. When present, IgG2 responses were of low titre but high functional affinity with no change in response to immunization and may represent a T-independent cross-reacting response with a carbohydrate epitope of KLH. IgG3 responses were variable but generally of low titre and low functional affinity, although those of higher titre and affinity were associated with secondary delayed-type hypersensitivity (DTH) and clinical reactions to KLH. In contrast to IgG1, IgG4 antibodies were not detected until 1 year after primary immunization, when they were found at low titre but high functional affinity. Following secondary immunization, IgG4 titres increased rapidly but without any further increase in affinity. The emergence of high-affinity IgG4 antibodies coincided with a loss of the high-affinity IgG1 populations, suggesting a preferential switch with time from high-affinity IgG1 antibodies to IgG4.