Kaposi's sarcoma associated herpesvirus (KSHV)/human herpesvirus 8 (HHV-8) encodes an immediate early transcriptional activator, Rta, which mediates viral reactivation from latency and lytic viral replication. Here we report the purification and characterizations of HHV-8 Rta and its interaction with Rta-responsive DNA elements. The Rta response element (RtaRE) in the promoter of the KSHV/HHV-8 K8 open reading frame was mapped to a 47-bp sequence (RtaRE1) and a 60-bp sequence (RtaRE2) upstream of the TATA motif. A comparison of the K8 RtaREs with other viral RtaREs revealed a pattern of multiple A/T triplets spaced with a periodicity of 10 or 20 bp. Substitutions of the in-phase A/T trinucleotides of the RtaRE1 with G/C bases greatly diminished Rta responsiveness and Rta binding. By contrast, base substitutions in an out-of-phase A/T-trinucleotide sequence had no effect. Importantly, multimers of (A/T)(3)N(7) and N(5)(A/T)(5)N(6)(A/T)(4) motifs supported a strong Rta response in a copy number-dependent manner. No specific sequence motifs in the spacer regions could be discerned. Potent Rta response, however, was obtained with phased A/T trinucleotides with 7-bp spacers of arbitrary sequences with high G/C content. Lengthening of the phased A/T motifs or lowering of the G/C content of the spacers resulted in a reduction in Rta response. Finally, Escherichia coli-derived Rta is an oligomer of 440 kDa in molecular size and binds RtaRE as an oligomer. These results support a model of Rta transactivation wherein the subunits of the Rta oligomer make multiple contacts with a tandem array of phased A/T triplets in the configuration of (A/T)(3)(G/C)(7) repeats.