Somatic hypermutation (SHM) is a hallmark of antibody-secreting B cells used to produce mature and high affinity antibodies. SHM takes place in the variable region and within the 1.5 kb downstream region of the immunoglobulin (Ig) gene promoter but no mutations have been thus far reported in the constant region. This study reports the characterization of DNA immunization-generated hybridoma clones secreting functional and Blo t 11-specific monoclonal antibodies but with mutations on the constant region of the Ig gene. The nucleotide sequence of the constant region of the Ig gene of four mutant hybridoma clones secreting Blo t 11-specific monoclonal antibodies that are total Ig-positive was characterized. The antibodies had no detectable isotype/subclass by enzyme-linked immunosorbent assay (ELISA). A series of transition, transversion, deletion and insertion mutations were detected on exons 3 and 4 but not on exons 1 and 2 of the Ig gene. Activation-induced cytidine deaminase (AID) transcripts were observed in all mutant hybridomas examined. Sequence analysis showed that 7 and 9 of the observed mutations in clones P1C9 and P1A6, respectively, were located on RGYW and WRCY hotspots where AID have been reported to occur at high frequencies. The results presented in this study may represent a novel observation related to Ig gene SHM and maturation that may be associated with DNA immunization.*To whom the correspondence should be addressed.