We have characterized a novel substrate for somatic hypermutation, confirming that non-Ig sequences can be targeted for mutation and demonstrating that this substrate allows for the rapid assay for mutations. An artificial sequence containing alternating EcoRV and PvuII sites (EPS) was inserted into the Vkappa167 transgene, which is known to be a target for mutation. To assay for somatic hypermutation, the EPS is amplified using flanking transgene primers, and the PCR product is subsequently digested with either EcoRV or PvuII. A mutation is seen as the appearance of a larger fragment, indicating a base change in a restriction enzyme site. The original transgene, Vkappa167/EPS, showed evidence of a low level of mutation in both splenic hybridomas and Peyer's patch-derived or immunized splenic B220+ cells with high peanut agglutinin levels. Two derivative lines of Vkappa167/EPS were made, Vkappa167/POX and Vkappa167/PEPS. While none of the Vkappa167/POX transgenic lines demonstrated mutation, the Vkappa167/PEPS transgene was highly mutated in B220+ splenic B cells with high peanut agglutinin levels at a frequency similar to that of endogenous Ig genes. An analysis of splenic RNA from the unimmunized transgenic mice indicated that the levels of stable message in splenic B cells could not be correlated with the mutation seen in GC B cells. The mutable Vkappa167/PEPS transgenic line is a unique tool to study somatic hypermutation in vivo.