Melanocytes express MITF, which is crucial for the development of the melanocyte lineage and is overexpressed in malignant melanomas. Adenoviral E1A protein-expressing melanocytes are unpigmented, with the expression of MITF being silenced. We tested here a direct repression of the melanocyte-specific MITF promoter by E1A and its mutants. We found that the extreme N-terminus and conserved region 1 are required for repression. In contrast, the motif in conserved region 2 (a.a. 122-126), as well as amino acids 26-35 at the N-terminus, are not necessary. As these two later motifs mediate E1A binding to the retinoblastoma protein or to the transcriptional co-activator TRRAP, respectively, and are important for transformation by E1A in cooperation with other oncogenes, the results suggest that the transformation-defective E1A can still efficiently repress the MITF promoter. The CREB binding motif-mutated promoter had lower activity, but was also repressed by the same E1A mutants in human melanoma cells. The E1A protein is known to also exert an antitumour activity, which is associated with its transcription repression function and the ability to induce apoptosis, and is a potential antimelanoma agent. Since recent data suggest that MITF may be a survival factor for melanoma cells, the E1A mutants described here might constitute a good targeting agent for antimelanoma therapy.