Krox proteins are important regulators of development and terminal differentiation. Using the rat glial progenitor cell line CG-4 as a model system for oligodendrocyte differentiation, we show that on the RNA level Krox-24 is the predominant member of the Krox family in these cells. Similar results were also obtained on the protein level as the major Krox protein from CG-4 cell extracts reacted specifically with an antibody against Krox-24. Whereas Krox-24 RNA and protein were abundant in undifferentiated CG-4 cells, a dramatic decrease in expression was detected after a 3-5-day period of differentiation during which we observed a reciprocal increase in the levels of myelin basic protein expression. Importantly, regulation of Krox-24 expression was very similar in CG-4 cells and primary oligodendrocyte cultures. When expression of Krox-24 in differentiating CG-4 cells was followed on a closer time scale, we observed a sharp and transient increase in Krox-24 RNA, protein, and DNA binding activity immediately after the onset of differentiation followed by an equally rapid decrease. This expression pattern implicates Krox-24 both in maintenance of the undifferentiated state and in the immediate early phase of differentiation of CG-4 cells and possibly oligodendrocytes.