Somatic hypermutation in rearranged immunoglobulin variable genes occurs in a 2kb region of DNA that is delimited on the 5' side by the promoter and on the 3' side by intron DNA. To identify sequence features that activate the mutation mechanism, we increased the distance between the promoter and the leader region to test whether the spacing of these elements was important. The promoter was separated from the leader sequence by inserting a 2 kb fragment of noncoding bacteriophage lambda DNA between the TATA box and ATG initiator codon in a kappa transgene. Mice from three founder lines were immunized, RNA and DNA were isolated from spleen and Peyer's patch B cells, and transcription of the transgene was confirmed. The frequency of mutation in endogenous heavy chain genes was high, indicating that some B cells underwent hypermutation. However, no hypermutation was found in the transgenic bacteriophage or variable region sequences. Hypermutation did occur in another kappa transgene that had a deletion of the VJ coding sequence, showing that the basic construct is functional and that the VJ exon is not necessary for the mutation mechanism. It is likely that the bacteriophage sequence is a potential substrate for mutation because other heterologous sequences have been shown to undergo mutation if placed downstream of the leader exon. The results suggest that the promoter should be contiguous with the leader exon for the mutation mechanism to function.