Specificity of phosphorylation is critical to signal transduction. Recent emphasis on colocalization of substrate and kinase has eclipsed emphasis on peptide specificity, i.e., kinase preference for particular amino acids surrounding the phosphorylation site. We describe an approach to determining peptide specificity by using positional scanning of biotinylated oriented peptide libraries and insights emerging from those determinations. We accurately determine preference (or disfavor) for residues at a given substrate position (such as P+2) by comparison of in vitro phosphorylation of peptide libraries differing by a single residue at that position. By analysis of all positions near the phosphorylation site, position-specific scoring matrices are generated and used both to understand the basis of specificity and to predict phosphorylation. PKC-delta and -zeta predictions have been validated rigorously by comparisons with measured phosphorylation. The results demonstrate specificity and sensitivity (80-90%) much better than the previous predictive method. These predictions can be accessed at http://mpr.nci.nih.gov. The accuracy of the specificity determination allows identification of an important difference in peptide specificity between these closely related kinases; Ile/Leu at the P-1 position is disfavored by PKC-zeta but not PKC-delta. Our findings and visual representation of peptide specificity highlight the importance of disfavored residues. Finally, analysis of 124 experimentally determined PKC sites from the literature demonstrates a very strong role of peptide specificity in many of those sites. Thus, position-specific scoring matrices generated by this method provide a foundation for quantitative analyses of kinase specificity and improved predictions of previously determined physiologically relevant phosphorylation sites.