Using an extensive series of deletion and site-specific mutation constructs, we have identified five new phosphorylation sites in c-Myc in the N-terminal transactivation domain and near the C-terminal DNA binding/heterodimerization domain. We have also found that Thr-58 phosphorylation is regulated by specific cellular events. When c-Myc is overexpressed in cells Thr-58 phosphorylation was greatly enhanced in the overexpressed, exogenous c-Myc as compared with the endogenous protein. In contrast, an inhibition of Thr-58 phosphorylation and an enhancement of Serine 62 phosphorylation was observed in c-Myc from immortalized cells compared with primary cells. No significant changes in c-Myc phosphorylation were found when transformed and nontransformed cells were compared. Finally, mutations at these phosphorylation sites, either individually or in combination with previously described sites, did not affect the ability of c-Myc to transactivate through the CACGTG Myc/Max DNA binding sites. These results further suggest that either the molecular role for c-Myc phosphorylation does not involve modulating transcriptional activity of c-Myc or that the CACGTG site does not represent a physiological promoter element.