3T3-L1 adipoblasts that express large amounts of c-Myc cannot terminally differentiate, raising the possibility that Myc inhibits the expression of genes that promote adipogenesis. The CCAAT/enhancer binding protein (C/EBP-alpha) is induced during 3T3-L1 adipogenesis when cells commit to the differentiation pathway. Transfection of 3T3-L1 adipoblasts with the gene that encodes C/EBP-alpha caused overt expression of the adipocyte morphology. Expression of Myc prohibited the normal induction of C/EBP-alpha and prevented adipogenesis. Enforced expression of C/EBP-alpha overcame the Myc-induced block to differentiation. These results provide a molecular basis for the regulation of adipogenesis and implicate Myc and C/EBP-alpha as pivotal controlling elements.