Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer in sheep. One of the unique features of this virus is that in infected animals, the only tissues that show expression of the virus are the tumor cells in the lung. We previously showed that the JSRV long terminal repeat (LTR) is preferentially active in murine lung epithelial cell lines (MLE-15 and mtCC1-2). To further explore the tissue specificity, we inserted the JSRV enhancer sequences from the U3 region of the LTR into a Moloney murine leukemia virus (M-MuLV) LTR lacking its own enhancer sequences, to give the chimeric LTR DeltaMo + JS. Transient transfection assays indicated that the DeltaMo + JS LTR is > 5-fold more active in lung epithelial cell lines than in non-lung lines, compared to the wild-type M-MuLV LTR. This was due to preferential activity of the JSRV enhancers in lung epithelial cells. Moreover, M-MuLV driven by the DeltaMo + JS LTR was > 3 logs more infectious in MLE-15 cells compared to non-lung cell lines. This chimeric virus may facilitate investigations of the tissue-specificity of JSRV.