To determine what genetic changes are selected in the enhancer sequences of the feline leukemia virus (FeLV) long terminal repeat in cats that develop T cell tumors, we cloned proviral U3 sequences in cats that died with thymic lymphoma following infection with molecularly cloned FeLV. Analysis of the U3 enhancer region revealed single base changes, including point mutations in the core and FLV-1 sequences. Additionally, in clones from two of four cat tumors, portions of the enhancer including Lvb and core were duplicated with respect to the single enhancer unit of the inoculating virus. In contrast, a PCR survey of necropsy DNA samples derived from five cats that did not develop tumors revealed that all retained the single enhancer unit of the infecting virus. These results demonstrate that viruses with duplicated enhancers can be generated and selected after only a single passage in cats, and furthermore, that such viruses may be particularly selected in tumors.