ABSTRACT MyoD is a nuclear phosphoprotein that belongs to the family of myogenic regulatory factors and acts in the tran-scriptional activation of muscle-specific genes. We have investigated the role of cAMP-dependent protein kinase (A-kinase) in modulating the nuclear locale of MyoD. Purified MyoD protein microinjected into the cytoplasm of rat embryo fibroblasts is rapidly translocated into the nucleus. Inhibition of A-kinase activity through injection of the specific inhibitory peptide PKI prevents this nuclear localisation. This inhibition of nuclear location is specifi-cally reversed by injection of purified A-kinase catalytic subunit, showing the requirement for A-kinase in the nuclear import of MyoD. Site-directed mutagenesis of all the putative sites for A-kinase-dependent phosphorylation on MyoD, substituting serine or threonine residues for the non-phosphorylatable amino acid alanine, had no effect on nuclear import of mutated MyoD. These data exclude the possibility that the effect of A-kinase on the nuclear translocation of MyoD is mediated by direct phosphoryla-tion of MyoD and imply that A-kinase operates through phosphorylation of components involved in the nuclear transport of MyoD.