Human glycophorin C (GPC) is an integral membrane protein found in many cell types but which exhibits increased expression in red cells. We have investigated the in vitro binding of nuclear protein factors to the GPC gene 5'-flanking region which was transcriptionally active when transfected into erythroid and non-erythroid cells. We found that in addition to known ubiquitous or erythroid trans-acting factors, a presently uncharacterized erythroid/megakaryocyte-specific protein binds the GPC promoter. This factor, that we called NFE-6, is present in embryonic, fetal, or adult-like human or mouse erythroleukemic cell lines, in a megakaryoblastic cell line, but not in lymphoid or non-hematopoietic cell lines. These data suggest that NFE-6, like the well characterized NFE-1 factor, is tissue- but not stage-specific and is conserved across species. It is proposed that NFE-6 might be involved in the quantitative change in the expression of the house-keeping GPC gene related to red cell specialization.