Transcriptional regulation by the ubiquitous human POU homeo domain protein Oct-1 and the related B-cell protein Oct-2 is a model for understanding how proteins that recognize the same regulatory site elicit different programs of gene transcription. Here, we describe a mechanism for differential promoter activation whereby only Oct-1, through selective corecruitment with the herpesvirus trans-activator VP16, acquires the ability to stimulate transcription from a TAATGARAT-containing site that responds to neither Oct-1 nor Oct-2 alone. To measure differential in vivo activation by human Oct-1 and Oct-2 in response to VP16, we have developed a transient assay in murine NIH-3T3 cells. Surprisingly, murine Oct-1 associates with VP16 much less effectively than its human counterpart, most likely because the murine Oct-1 homeo domain differs at four positions from the human Oct-1 homeo domain. The murine cell transient assay shows directly that human Oct-1, but not human Oct-2, can respond to VP16 in vivo. The Oct-1 DNA-binding POU domain is sufficient and the Oct-1 homeo domain is critical for this response, because an Oct-1 POU domain containing the Oct-2 homeo domain fails to respond to the VP16-induced positive control of transcription. Thus, by selective homeo domain interaction and corecruitment to an otherwise silent regulatory element, VP16 expands the repertoire of sites responsive to Oct-1 without affecting the activity of its close relative Oct-2.