The mechanism of somatic hypermutation in the variable region of immunoglobulin genes expressed in mammalian B cells is a major unexplained phenomenon in the generation of diversity in the immune system. To evaluate possible mechanisms, the distribution of somatic mutations was examined for a group of five cloned, rearranged, somatically mutated VH genes generated in C57BL/6j mice. These mutated VH genes were sequenced and compared with their germ line counterparts from a point approximately 550 base pairs upstream of the transcription start site to an EcoRI site some 1,200 base pairs downstream of JH-4. The location of the transcription start (cap) sites was also precisely determined. Most (greater than or equal to 94%) of the 118 mutations scored occurred between the transcription start site and the distal end of JH-4. However, seven mutations occurred upstream of the transcribed region, and at least four were found downstream of JH-4. The target region for the mutator mechanism therefore clearly extends into the 3' nontranslated and 5' nontranscribed regions. Thus, models which propose the transcribed region of the DNA as the sole substrate for the mutation process are not ruled out but are inadequate to explain the upstream distribution of somatic mutations.