The c-myb proto-oncogene encodes a nuclear protein involved in the regulation of cell proliferation, differentiation, and development. Myb protein contains a DNA binding and a transactivating domain thought to mediate its biologic properties. The DNA binding domain consists of three repeats (R1, R2, and R3), each containing a highly conserved motif of tryptophan residues. A c-myb mutant (DR1-myb) lacking the last 46 amino acids of R1 and 23 amino terminal residues of R2, a region homologous to the ADA-2 yeast transcriptional adaptor, lost DNA binding ability, but remained able to transactivate the human heat-shock promoter. Transfection of murine 32D and murine erythroleukemia (MEL) cell lines with DR1-myb caused inhibition of cellular differentiation induced by granulocyte colony-stimulating factor (G-CSF) and dimethyl sulfoxide (DMSO), respectively. A second c-myb mutant (D-ADA2-myb) lacking the first 23 amino acids of R2, also lost DNA binding and transactivation activity, but did not inhibit DMSO-induced differentiation of MEL transfected cells. These findings suggest that deletion of R1 activates a DNA binding-independent mechanism of c-myb function, which may involve interaction of Myb with cellular factors.