Epstein-Barr virus, a human gammaherpesvirus, possesses a unique set of latent genes whose constitutive expression in B cells leads to cell growth transformation. The initiation of this growth transforming infection depends on a viral promoter in BamHI W (Wp) whose regulation is poorly understood. Using Wp reporter constructs in in vitro transfection assays, we found that Wp was 11- to 190-fold more active in B cell than in non-B cell lines and that three regions of the promoter (termed UAS1, UAS2, and UAS3) contributed to transcriptional activation. The upstream regions UAS3 (-1168 to -440) and UAS2 (-352 to -264) both functioned in a cell lineage-independent manner and were together responsible for the bulk of Wp activity in non-B cells; mutational analysis indicated the importance of a YY1 binding site in UAS2 in that context. By contrast, UAS1 (-140 to -87) was B cell specific and was the key determinant of the promoter's increased activity in B cell lines. Mutational analysis of UAS1 sequences combined with in vitro bandshift assays revealed the presence of three binding sites for cellular factors in this region. When mutations that abolished factor binding in bandshift assays were introduced into a Wp reporter construct, the loss of any one of the three UAS1 binding sites was sufficient to reduce promoter activity by 10- to 30-fold in B cells. From sequence analysis, two of these appear to be novel transcription factor binding sites, whereas the third was identified as a cyclic AMP response element (CRE). Our data indicate that this CRE interacts with CREB and ATF1 proteins present in B cell nuclear extracts and that this interaction is important for Wp activity.