c-Myc is a nuclear phosphoprotein which binds DNA as a heterodimer with Max. We have identified two in vivo phosphorylation sites, Thr58 and Ser62, within a domain highly conserved among all Myc family members. Thr58 is mutated in several viral forms of the protein and constitutes a mutational hot-spot in Burkitt's lymphoma. Members of the GSK-3 and MAP kinase families, but not CKII, specifically phosphorylated these sites in vitro. The effect of these phosphorylation sites on Myc function was assessed by cotransformation of primary rat embryo fibroblasts with Ras. Mutagenesis of Thr58 to alanine potentiated focus formation, whereas substitution of Ser62 severely inhibited transformation. Mutation of both residues restored wild-type activity. These data suggest acute, post-translational modulation of Myc via phosphorylation of a conserved region previously implicated in transactivation, transformation and autorepression.