A sensitive exonuclease assay revealed multiple sites for interaction, in vitro, of sequence specific factors with c-myc upstream elements. At one site, more than 1500 base pairs upstream of the c-myc promoter P1, binding activity was lost as dimethyl sulfoxide (Me2SO) induced shut-off of c-myc expression in HL-60 and U-937 leukemia cells. The disappearance of other specific binding activities was not noted. In addition, the binding activity was noted to be cell-line specific. The sequence binding the Me2SO-regulated factor was precisely located allowing confirmation of the temporal pattern of regulation by electrophoretic mobility shift analysis. Because the binding activity was most abundant before the decrease of c-myc expression during differentiation, it was inferred that the far upstream element (FUSE) served a positive role, potentiating c-myc expression. A 4-base pair deletion which eliminated binding to FUSE also reduced expression of a transfected, chimeric c-myc-CAT gene in untreated, but not in Me2SO-treated U-937 cells. FUSE and its binding protein may contribute to cell line- and differentiation-specific modes of c-myc regulation.