Human spleen immunoglobulin gene rearrangements that used the VH6 gene and were expressed with IgM were characterized for their frequency of somatic hypermutation from PCR amplified spleen cDNA. A high frequency of rearrangements that were somatically mutated was demonstrated by restriction endonuclease analysis and sequencing of cloned rearrangements. The 24 rearrangements cloned from three different spleens had an overall mutation frequency of 3.1% mutations/bp sequenced and ranged from 0.4 to 6.0%. These mutations appeared to have been antigenically selected based on both the high frequency and high amino acid replacement to silent (R/S) ratios in the complementarity determining regions. Five clones that arose from two different rearrangements showed evidence of intraclonal diversification with both shared and unique mutations. The mutated clones of one spleen donor were lower in frequency and were not concentrated in the CDR, which suggested these mutations had not been antigenically selected. These findings support the dissociation of somatic mutation and isotype switching and the possibility that IgM-expressing B cells may serve as human memory B cells.