The site and sequence specificity of protein kinases, as well as the role of the secondary structure and surface accessibility of the phosphorylation sites on substrate proteins, was statistically analyzed. The experimental data were collected from the literature and are available on the World Wide Web at http://www.cbs.dtu.dk/databases/PhosphoBase/. The set of data involved 1008 phosphorylatable sites in 406 proteins, which were phosphorylated by 58 protein kinases. It was found that there exists almost absolute Ser/Thr or Tyr specificity, with rare exceptions. The sequence specificity determinants were less strict and were located between positions -4 and +4 relative to the phosphorylation site. Secondary structure and surface accessibility predictions revealed that most of the phosphorylation sites were located on the surface of the target proteins.