The VH5 family contains two functional genes, V5–51 and VH32, and appears to be over-represented in IgE antibodies from patients with allergic disease. Previous sequence analysis of VH5 gene segments in IgE has revealed a substantial level of somatic hypermutation, with evidence for hotspots. To assess characteristics of VH5 gene behavior, VH5 gene segments in combination with Cμ, Cγ, Cα, and Cε have been amplified from blood B lymphocytes of a patient with atopic asthma. Sequence analysis revealed strong preferential usage of one of the two VH5 gene segments (V5–51) by IgM, IgG, and IgA. In contrast, IgE used both genes equally. Levels of somatic mutation were higher following all isotype switches, particularly to IgA. Mutational hotspots were identifiable in all isotypes, leading to several common replacement amino acids. The dominant mutational site in IgM was a common hotspot at Ser31. IgG, IgA, and IgE-derived sequences had mainly common hotspots, with few distinct sites. The results indicate that mutational hotspots are a feature of the VH5 gene, are identifiable at an early stage of somatic hypermutation, and are not a unique feature of IgE. Generation of IgE antibodies appears to involve three processes: the preferential use of VH5 genes, consistent with superantigen stimulation; the accumulation of somatic mutations in common hotspots, some of which are in complementarity-determining regions (CDR); and the acquisition of non-hotspot mutations in CDR, accounting for ∼ 50% of replacement amino acids in these sites, and presumably contributing to affinity maturation.