Mice with a homozygous inactivation of the transcription factor gene Spi-B(-)/(-) have abnormal B cell functions and a defect in germinal center (GC) formation. We report here that somatic hypermutation (SHM) of VH1 and VH11 genes is not diminished in Peyer's patches of Spi-B(-)/(-) mice. However, the mutation pattern shows an increase in the ratio of replacement to silent mutations in the framework sequences of the variable regions, suggesting that selection of mutated B cells based on functionality is affected. In support of this, two of the six sequences from Spi-B mutant mice have a point mutation in the framework which introduces predicted steric clashes with another amino acid in the variable region. This mutation (Leu81Phe) has not been observed in 120 mutated VH1 or VH11 genes of germinal center B cells from Spi-B wildtype mice. The mutation also does not exist in any of 136 published heavy chain proteins of the same VH family. The mutations causing the change to Phe are transitions which are favored by the SHM process over transversions. Clearly, Phe-81 must arise relatively frequently, but is not selected in Spi-B wildtype mice.