SL3-3 is a highly T-lymphomagenic murine retrovirus in which the transcriptional enhancer is a major oncogenic determinant. Here, we describe an SL3-3 enhancer variant that induced T-cell lymphomas in all inoculated mice with a shorter latency period than wild-type SL3-3. The enhancer repeat region of this variant contains two deletions encompassing the nuclear factor 1 binding sites in addition to an additional intact enhancer repeat element. Tumors induced by this variant were T-cell lymphomas, as indicated by T-cell receptor rearrangements, and contained the input provirus enhancer regions. The variant was the result of mutation of specific transcription factor binding sites in the viral enhancer, isolation of rare second-site enhancer variants from the resulting induced tumors, and subsequent restoration of the original first-site mutations of one such variant. We have termed this process assisted molecular evolution.