We examined acetylation of the histone-like adenovirus core proteins VII and V and the precursor of the major core protein, pVII, by measuring the incorporation of [14C]acetate. Adenovirus proteins pVII and V appeared to be acetylated, whereas protein VII was not. Label incorporated into these viral proteins in the form of acetate was metabolically stable, and labeling was not enhanced by treatment with sodium butyrate, an inhibitor of histone deacetylases. Viral protein acetylation therefore differs from the reversible acetylation of histones that has been implicated in transient alterations of chromatin structure. Inhibition of protein synthesis in infected cells resulted in a proportional reduction in [14C]acetate uptake into pVII and V, suggesting that these proteins undergo acetylation during protein synthesis and not as a post-translational modification. Therefore, these viral proteins are probably acetylated amino-terminally, a characteristic shared by three of the five major histone classes.