The histo-blood group i and I antigens have been characterized as straight and branched repeats of N-acetyllactosamine, respectively, and the conversion of the straight-chain i to the branched-chain I structure on red cells is regulated to occur after birth. It has been demonstrated that the human I locus expresses 3 IGnT transcripts, IGnTA, IGnTB, and IGnTC, and that the last of these is responsible for the I branching formation on red cells. In the present investigation, the K-562 cell line was used as a model to show that the i-to-I transition in erythroid differentiation is determined by the transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha), which enhances transcription of the IGnTC gene, consequently leading to formation of the I antigen. Further investigation suggested that C/EBPalpha IGnTC-activation activity is modulated at a posttranslational level, and that the phosphorylation status of C/EBPalpha may have a crucial effect. Results from studies using adult and cord erythropoietic cells agreed with those derived using the K-562 cell model, with lentiviral expression of C/EBPalpha in CD34(+) hemopoietic cells demonstrating the determining role of C/EBPalpha in the induction of the IGnTC gene as well as in I antigen expression.