PC4 (pheochromocytoma cell-4) is an immediate early gene related to IFN-gamma, the mRNA of which is induced during the course of neuronal differentiation by nerve growth factor in the PC12 cell line. Here we report that PC4 mRNA is also expressed in the myoblast C2C12 cell line and is regulated during differentiation; its expression decreases within 6 h from the onset of differentiation, attains a minimum after 12 h, and returns to basal level within 36 h. This transient down-regulation of PC4 expression in C2C12 myoblasts is prevented by transforming growth factor beta, a molecule which inhibits the differentiation of muscle. Sense and antisense PC4 cDNA transfection strategies in C2C12 cells were then used to clarify the role of PC4 in muscle differentiation. While no effect was seen by over-expression of PC4, stable transfectants underexpressing PC4 exhibited a delay in attaining the differentiated phenotype, with an impairment of myogenin and myosin expression. Myogenin was also inhibited in C2C12 cells microinjected with the anti-PC4 polyclonal antibody A451. We thus postulate a role for PC4 as a positive regulator during muscle differentiation.