This study examines the role of enhancer elements in the expression of the Prague strain of Rous sarcoma virus, PrRSV. Viral enhancer elements have been shown to promote high level expression of viral genes as well as cellular genes adjacent to the integrated proviral DNA. Previous studies have associated the 3' LTR and immediate upstream sequences (called the XSR) with the disease spectrum of the tumor defective (td) variants of PrRSV. It is the intent of this study to identify and characterize the enhancer elements in this region and to correlate their properties with the disease spectrum of the virus. One enhancer element has been identified which contains 90 nucleotides of the 3' LTR as well as adjoining upstream sequences. This enhancer exhibits a host preference for expression in chicken cells. An additional enhancer sequence is found entirely within the LTR. We suggest that multiple enhancer domains are present in the 3' terminus of PrRSV and that the presence of at least two of these domains is required for enhancer function.