A negative regulatory element in the 5'-flanking region of the murine glut4 gene mediates chronic insulin- and cAMP-induced repression in 3T3-L1 adipocytes. Previous work demonstrated that members of the nuclear factor 1 (NF1) family of transcription factors and an unidentified factor bind to and mediate repression from this regulatory element. By using a yeast one-hybrid screen, Olf-1/Early B cell factor (O/E-1) was isolated as a candidate for this unidentified factor. A protein complex from 3T3-L1 adipocyte nuclear extract that bound the negative regulatory element was recognized by O/E-specific antiserum, and binding activity was competed effectively by distinct O/E-binding sequences. O/E binding activity was also detected in nuclear extracts from insulin-responsive, GLUT4-expressing tissues including adipose, skeletal muscle, and heart. Mutations within the negative regulatory element that abolish binding of O/E proteins concomitantly blocked insulin-induced repression in reporter gene assays. These results suggest that one or more members of the O/E transcription factor family function as important regulators of glut4 gene expression and therefore may play a heretofore unanticipated role in glucose homeostasis and insulin signaling.