Lentiviruses vary in their dependence on a functional tat gene during their viral life cycle. To begin to understand the viral and cellular parameters controlling equine infectious anemia virus (EIAV) transactivation, we investigated Tat function and Tat and LTR structural requirements necessary for successful transactivation. EIAV Tat expression was required for detection of viral antigens from a full-length provirus. The level of transactivation by EIAV Tat as measured by LTR-CAT assays correlated well with viral antigen expression. Using horse/mouse somatic cell hybrids (SCH), a single SCH line which supported EIAV transactivation was identified, indicating that the presence of specific horse chromosomes provided cellular factors required for transactivation. Transformed cell lines from several different species were also tested and found to differ in their ability to support EIAV transactivation. A canine cell line, Cf2Th, which was permissive for EIAV transactivation, and a human cell line, HeLa, which was not permissive for EIAV transactivation, were used to map regions of the LTR and Tat that were important in cell-specific transactivation. As expected, the R region of EIAV LTR was required for transactivation by EIAV Tat in all cell lines studied. Similarly, the R region of HIV LTR was necessary for transactivation by HIV Tat. However, the composition of the U3 region also influenced transactivation in a cell-specific manner. In Cf2Th cells, replacement of EIAV U3 sequences with HIV U3 sequences resulted in high basal (nontransactivated) expression, and as a result, only a twofold increase in expression was observed in the presence of EIAV Tat. Similar studies using HIV Tat demonstrated that transactivation occurred in Cf2Th cells when either EIAV or HIV U3 sequences were present in the LTR. In contrast, transactivation by either HIV or EIAV Tat in HeLa cells required the presence of HIV enhancer sequences. These findings suggested that the ability of transactivation to occur in some cell lines may involve interactions between cell-specific transcription factors and the activation domain of Tat. For transactivation in other cell lines, Tat appeared to require more ubiquitious factors that interact with both EIAV and HIV U3 sequences.