Marek's disease virus transactivates promoters of avian leukemia and sarcoma viruses. In this study, a series of RSV-LTR promoter deletion mutants were used to map sites within the LTR important for MDV-mediated transactivation. MDV-responsive elements within RSV-LTR promoters were localized to a 28-bp segment (nucleotides -109 to -137) which contains a GGTGG pentanucleotide repeat element (PRE). Nuclear extract proteins from uninfected cells bound to the RSV PRE in a sequence-specific manner. Extracts from cells infected with MDV produced novel, sequence-specific complexes with RSV PRE probes. Transactivation in other herpesvirus-retrovirus systems has been shown to depend, at least in part, on expression of herpesvirus immediate-early genes. In this report, we demonstrate that MDV ICP4 is capable of transactivating RSV-LTR promoters containing an intact PRE region. Transactivation with an isolated MDV ICP4 gene expressed from its cognate promoter was less efficient than with intact MDV, suggesting that other MDV-encoded factors are likely to play a role in MDV-mediated transactivation of RSV-LTR promoters. RSV-LTR promoters lacking a PRE region were not efficiently transactivated by MDV ICP4. We conclude that MDV ICP4 may be at least partially responsible for transactivation of RSV-LTR promoters and that this transactivation is likely to be dependent upon presence of a PRE region within RSV-LTR promoters.