Epstein-Barr virus (EBV) latent-infection membrane protein (LMP) gene cis-acting regulatory sequences were assayed in human B lymphocytes by using chloramphenicol acetyltransferase (cat) gene expression as a reporter. The activities of progressively longer upstream elements from bases -55 to -2350 were compared. At least two positive cis-activating regulatory components (-155 to -147 and -234 to -205) upstream of the LMP promoter were defined. LMP promoter cat gene constructs were more active in a Burkitt's lymphoma cell line latently infected with the B95 EBV strain than in the same cells latently infected with the P3HR1 EBV strain. Since the P3HR1- and B95-infected cells differ in EBNA-2 and EBNA-LP expression, EBNA-2 or EBNA-LP is a likely transactivator of the LMP promoter. Probable cognate sequences for known transcription factors in the LMP promoter are discussed.