The effects of cyclic AMP-dependent protein kinase (cAMP-PK) or Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation on the binding of bovine tau to tubulin and calpain-mediated degradation of tau were studied. Both cAMP-PK and CaMKII readily phosphorylated tau and slowed the migration of tau on sodium dodecyl sulfate-containing polyacrylamide gels. However, cAMP-PK phosphorylated tau to a significantly greater extent than CaMKII (1.5 and 0.9 mol of 32P/mol of tau, respectively), and phosphorylation of tau by cAMP-PK resulted in a greater shift to a more acidic, less heterogeneous pattern on two-dimensional nonequilibrium pH gradient gels compared with CaMKII phosphorylation. Two-dimensional phosphopeptide maps indicate that cAMP-PK phosphorylates a site or sites on tau that are phosphorylated by CaMKII, as well as a unique site or sites that are not phosphorylated by CaMKII. Phosphorylation of tau by cAMP-PK significantly decreased tubulin binding and, as previously reported, also inhibited the calpain-induced degradation of tau. CaMKII phosphorylation of tau did not alter either of these parameters. These results suggest that the phosphorylation of site(s) on the tau molecule uniquely accessible to cAMP-PK contributed to the decreased tau-tubulin binding and increased resistance to calpain hydrolysis.