Unlike most Ser/Thr protein kinases which recognize phosphoacceptor sites specified by basic residues, protein kinase CK2 is extraordinarily acidophilic in nature. By combining the analysis of more than 100 CK2 natural phosphorylation sites with the kinetic behaviour of a large number of model peptide substrates, it can be concluded that although the most crucial specificity determinant is an acidic residue (Glu, Asp, TyrP, or SerP) at position +3, additional acidic residues at positions spanning from -2 to +7 (and probably farther) also act as positive specificity determinants for CK2, whereas basic residues at these positions, prolyl residue at position +1, and a bulky hydrophobic doublet at position +1 and +2, are powerful negative determinants. It also appears that the nature of the acidic determinants may variably influence their effect depending on the position occupied: Thus, multiple aspartic acids are, in general, determinants as good as, or even better, than an equivalent number of glutamic acids; an individual Asp at position +3 flanked by Glu residues is ineffective; and phosphorylated residues appear to be much more effective if adjacent to the target residue (positions -2 to +2). In some instances, the local determinants alone are insufficient to account for the phosphorylation efficiency of the substrate which is greatly improved by the overall protein conformation, as illustrated by the examples of CK2 beta-subunit and protein p53, the latter exhibiting no consensus sequence around its phosphorylation site.(ABSTRACT TRUNCATED AT 250 WORDS)