Recombination studies have established that retroviral long terminal repeats (LTRs) are important genetic determinants of the viral capacity to induce hematopoietic tumors and to specify the type of cell making up the tumor. Plasmids containing LTRs of several murine leukemia viruses linked to the chloramphenicol acetyltransferase gene were tested in transient assays to measure relative rates of transcriptional activity in different types of hematopoietic cells. LTRs of the thymomagenic viruses SL3-3, Moloney leukemia virus, and a Moloney mink cell focus-forming virus all expressed to higher levels than other LTRs in T-lymphocyte cell lines. Conversely, the LTRs of Friend leukemia virus and a polycythemic spleen focus-forming virus expressed to higher levels than other LTRs in erythroleukemia cells. The LTR of nonleukemogenic Akv virus induced a relatively low level of activity compared with the others in all cells tested. Thus the relative level of LTR-driven expression in various types of cells corresponds to the type of tumor caused by the intact virus in vivo. These results provide direct evidence that the tissue specificity of the transcriptional activity of LTRs plays a critical role in determining the target cell for retroviral oncogenesis.