The human herpes simplex virus type 1 (HSV-1) transactivator VP16 and its homolog from bovine herpes-virus 1 (BHV-1) can each recruit the human homeodomain protein Oct-1 into a transcriptional regulatory complex. Here, we show that these two Oct-1 coregulators possess similar, if not identical, homeodomain recognition properties but possess different virus-specific cis-regulatory specificities: the HSV-1 VP-16 protein activates transcription from the HSV-1 VP16 response element, and the BHV-1 VP16 protein activates transcription from the BHV-1 VP16 response element. A distinct 3-bp segment, the D segment, lying 3' of the canonical TAATGARAT motif (where R is a purine) in the VP16 response element is responsible for the differential cis element recognition and transcriptional activation by these two homeodomain coregulators. These results demonstrate how a single homeodomain protein can direct differential transcriptional regulation by selective association with homologous homeodomain coregulators.