Activation-induced cytosine deaminase preferentially deaminates C in DNA on the nontranscribed strand in vitro, which theoretically should produce a large increase in mutations of C during hypermutation of immunoglobulin genes. However, a bias for C mutations has not been observed among the mutations in variable genes. Therefore, we examined mutations in the mu and gamma switch regions, which can form stable secondary structures, to look for C mutations. To further simplify the pattern, mutations were studied in the absence of DNA polymerase (pol) eta, which may produce substitutions of nucleotides downstream of C. DNA from lymphocytes of patients with xeroderma pigmentosum variant (XP-V) disease, whose polymerase eta is defective, had the same frequency of switching to all four gamma isotypes and hypermutation in mu-gamma switch sites (0.5% mutations per basepair) as control subjects. There were fewer mutations of A and T bases in the XP-V clones, similar to variable gene mutations from these patients, which confirms that polymerase eta produces substitutions opposite A and T. Most importantly, the absence of polymerase eta revealed an increase in C mutations on the nontranscribed strand. This data shows for the first time that C is preferentially mutated in vivo and pol eta generates hypermutation in the mu and gamma switch regions.