Variable (V) genes of immunoglobulins undergo somatic hypermutation by activation-induced deaminase (AID) to generate amino acid substitutions that encode antibodies with increased affinity for antigen. Hypermutation is restricted to germinal center B cells and cannot be recapitulated in ex vivo-activated splenic cells, even though the latter express high levels of AID. This suggests that there is a specific feature of antigen activation in germinal centers that recruits AID to V genes which is absent in mitogen-activated cultured cells. Using two Igh knock-in mouse models, we found that RNA polymerase II accumulates in V regions in B cells after both types of stimulation for an extended distance of 1.2 kb from the TATA box. The paused polymerases generate abundant single-strand DNA targets for AID. However, there is a distinct accumulation of the initiating form of polymerase, along with the transcription cofactor Spt5 and AID, in the V region from germinal center cells, which is totally absent in cultured cells. These data support a model where mutations are prevalent in germinal center cells, but not in ex vivo cells, because the initiating form of polymerase is retained, which affects Spt5 and AID recruitment.