The hepatitis B virus (HBV) enhancer contains multiple active elements, one of which is the EP element, a 15 b site important for its regulation by acting on other functional elements like the E site. The EP element, in the HBV enhancer context, contains two putative binding sites for c-myb family gene products. Electrophoretic mobility shift assays showed that the minimal c-Myb DNA-binding domain binds to the EP sequence. DNase I footprinting experiments revealed that only one consensus binding site was effectively protected. We found that c-Myb down-regulates transcription driving by the HBV enhancer in CAT assays performed ina haematopoietic (K562) and in a hepatic (HepG2) cell line. Interestingly,coexpression of both c-Myb and NF-M, a C/EBP beta homologue which recognises the E element of the HBV enhancer, showed a synergistic transactivation of the HBV enhancer while, separately, each of them had an inhibitory effect on transcription ill HepG2 and K562 cell lines, two cell types potentially infected by the hepatitis B virus. (C) 1999 Elsevier Science B.V. All rights reserved.