Sixteen derivatives of the optimal peptide substrate RRRA-DDSDDDDD in which aspartic acids were singly or multiply substituted by alanine have been assayed for their phosphorylation efficiency by either wild type protein kinase CK2 or CK2 alpha mutants defective in substrate recognition. With wild type CK2, the only detrimental single substitutions were those at positions +3 and +1. Each of these caused a 5-fold increase of Km and a 2-fold decrease of the Vmax values. If both aspartic acids at n + 1 and n + 3 were substituted however, the Km rose 24-fold and the Vmax decreased 16-fold. Multiple substitutions tend to have a more than additive effect even if they affect individually dispensable aspartic acids; thus, double, triple, and quintuple substitutions at positions n - 2 and -1, and n + 2, +4, and +5 had detrimental consequences comparable to those observed with substitutions at n + 1 and n + 3. These data indicate that additional acidic residues besides those at n + 1 and n + 3 are collectively required for efficient phosphorylation of CK2 substrates. They are also consistent with a flexible mode of binding of the substrate, where acidic residues may play interchangeable roles. Among twelve CK2 mutants in which basic residues suspected to be implicated in substrate recognition have been replaced by alanine, only K74-77A, K79R80K83A, R191,195K198A, and K198A showed substantially increased Km values with the optimal substrate RRRA-DDSDDDDD, symptomatic of a reduced ability to bind it. However, if the suboptimal substrate RRRA-AASDDDDD was used, the single mutants K49A, K71A, K77A, R80A, and H160A also exhibited Km values significantly higher than those of wild type CK2. Kinetic analysis with singly substituted derivatives of peptide RRRA-DDSDDDDD revealed that K49 is implicated in the recognition of the determinant at position n + 2, K77 cooperates with other residues nearby in the interaction with the determinants at n + 3 and n + 4, while K198 plays a prominent role in the recognition of the determinant at n + 1.