Activation of the human transferrin receptor promoter by mitogenic stimulation of quiescent cells is a delayed event that reaches a maximum several hours after stimulation. Previous results have defined a region of the transferrin receptor gene promoter that is required for increased expression in mitogen-activated cells (W. K. Miskimins and D. B. Brown, Exp. Cell Res., 191: 328-331, 1990; Q. Ouyang et al., Mol. Cell. Biol., 13: 1796-1804, 1993). This region contains two elements (elements A and B) that appear to cooperate in the response to mitogenic stimulation. Serum stimulation of quiescent cells leads to the induction of nuclear factors that bind to both the A and B elements. Induction of these factors is also a delayed response to serum stimulation and reaches a maximum 6-9 h after stimulation. Element A, which is an unusual GC-rich sequence, forms several serum-inducible DNA-protein complexes, all of which depend on contacts within GC boxes. A major inducible complex of element A contains a factor that is supershifted by antibodies against the transcription factor Sp1. The B element appears to have overlapping binding sites for two types of factors. One of these sites binds factors that are competed off by an AP-1 consensus-binding site. The other B element site binds inducible factors that interact with GC boxes, identical to those observed for element A.