Cis-acting elements of the gene for mouse glycophorin, an erythroid-specific membrane glycoprotein, were determined by transient and stable transfection assays using murine erythroleukemia (MEL) cells. Cis-acting elements proximal to the transcription start site of the gene can be separated into the basal promoter (-1 to 191 bp) and the distal element (-133 to -92). The basal promoter contained GGTGG and GATA motifs and the distal element contained GATA-1 and NF-E2 motifs. Deletion analysis of the distal GATA site and its neighboring sequence and DNase-I footprinting/EMSA (electrophoretic mobility shift assay) analysis indicated that induced nuclear factor binding to GATA-1 and its neighboring sequence may be required for expression during MEL cell differentiation induced by dimethyl sulfoxide treatment. The NF-E2 site was also shown to be essential for the promoter activity. An approximately 400 bp far upstream region (-1325 to -948bp) containing the binding motifs for GGGTGG, GATA-1 and NF-E2 showed no enhancing activity when this region was examined by transient transfection assay, but it did show enhancement of the differentiation-specific promoter activity in the stable transfection assay. The far upstream region of mouse glycophorin gene may have a function similar to that of the locus control region (LCR) of human beta-globulin gene cluster.