Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus and an important human pathogen. Initiation of the EBV lytic cycle is dependent upon transcription of the EBV BZLF1 gene. Our previous studies of transcriptional regulation of the BZLF1 Z promoter (Zp) in human SCC12F epithelial cells identified a region within Zp that is responsive to epithelial cell differentiation. In the present study, we localize this differentiation responsive element to the CREB/AP-1-like binding site (TGACATCA) between -67 to -60 bp within Zp, previously designated ZII, and furthermore show that homodimers and heterodimers of CREB and ATF-1 specifically bind ZII. Consistent with a regulatory role for CREB and ATF-1 in differentiation dependent BZLF1 expression, ZII was able to bind approximately 3-fold more CREB and ATF-1 when incubated with nuclear extract obtained from populations of SCC12F cells enriched for the differentiated phenotype than when incubated with extract obtained from populations enriched for the undifferentiated phenotype. In addition, CREB and ATF-1 were found to increase in abundance during SCC12F differentiation. These results indicate a regulatory role for CREB and ATF-1 in differentiation-dependent expression of BZLF1 in human epithelial cells.