When human promyelocytic leukemia cell line HL60 was treated with retinoic acid (RA), considerable suppression of protooncogene myc expression was achieved before granulocytic differentiation became evident. From transient transfection experiments using the reporter plasmid containing exon 1 and its 2.3 kilobases upstream of the c-myc gene fused to the chloramphenicol acetyltransferase gene, it was indicated that this suppression was mainly attributable to the level of transcription initiation. Deletion down to 95 base pairs upstream of the P2 promoter did not change the suppressive effect of RA on c-myc gene expression. Mobility shift assays with respect to the P2 promoter region revealed that the 15-base pair fragment located between P1 and P2 promoters was responsive to the RA treatment. This fragment included the E2F binding site in the c-myc P2 promoter region, and a difference of shifted bands between RA-treated and untreated HL60 cells was due to complex formation of E2F and retinoblastoma protein. The present results suggest that E2F plays an important role in the process of cell differentiation by RA and that a change of the E2F binding pattern induced by RA contributes to the suppression of c-myc gene expression preceding granulocytic differentiation.