The detailed mechanisms leading to transcriptional activation of the human MYC oncogene in general as well as in certain tumor cells are poorly understood. In view of the ability of a number of viral oncogenes to stimulate transcription in trans, the identification of cellular target genes could contribute to the understanding of components of the transformation process. It is demonstrated that the human MYC promoter is such an efficient target for the trans-acting activity mediated by E1a proteins of adenoviruses (Ad). Using the chloramphenicol acetyltransferase (CAT) gene as a marker for promoter activity, co-transfection of constructs containing both MYC promoters and most of the untranslated first exon with plasmids expressing the E1a gene of different adenoviruses stimulates CAT activity up to 24-fold. Trans-activation depends upon the presence of the second promoter (P2), and transcription is initiated at the authentic cap site of P2. This observation is confirmed by the behaviour of stably transformed cell lines carrying single or multiple copies of MYC-cat constructs, which were transfected either with E1a-expressing plasmids, infected with Ad5, or fused with 293 cells constitutively expressing E1a protein. These results suggest that E1a proteins can lead to an imbalance of the regulation of the human MYC gene, which might be a sufficient prerequisite for initiation and progression of transformation.