We have found that the cAMP-dependent protein kinase catalyzes the phosphorylation of a wide variety of peptide-based aromatic alcohols, thereby greatly amplifying the range of compounds recognized as substrates by this enzyme. This newly discovered enzyme-catalyzed reaction is sensitive to both steric and electronic effects. Substituents on the aromatic ring that are positioned para to the hydroxyl moiety lower the observed Km, presumably via a favorable interaction with an adjacent hydrophobic pocket. In contrast, electron-withdrawing substituents have a slight adverse effect on the kinetics of phosphoryl transfer, an observation which is consistent with the notion that the rate of substrate turnover is dependent upon the nucleophilicity of the phosphorylatable hydroxyl moiety. As a corollary, electron-donating groups on the aromatic nucleus promote the rate of phosphoryl transfer to such an extent that the observed Vmax values approach those exhibited by aliphatic alcohols. This suggests that analogously appended electron-donating groups on tyrosine moieties could dramatically improve the modest Vmax values that are typical for tyrosine kinase-catalyzed reactions.