Tau in Alzheimer disease brain is highly phosphorylated and aggregated into paired helical filaments comprising characteristic neurofibrillary tangles. Here we have analyzed insoluble Tau (PHF-tau) extracted from Alzheimer brain by mass spectrometry and identified 11 novel phosphorylation sites, 10 of which were assigned unambiguously to specific amino acid residues. This brings the number of directly identified sites in PHF-tau to 39, with an additional six sites indicated by reactivity with phosphospecific antibodies to Tau. We also identified five new phosphorylation sites in soluble Tau from control adult human brain, bringing the total number of reported sites to nine. To assess which kinases might be responsible for Tau phosphorylation, we used mass spectrometry to determine which sites were phosphorylated in vitro by several kinases. Casein kinase 1delta and glycogen synthase kinase-3beta were each found to phosphorylate numerous sites, and each kinase phosphorylated at least 15 sites that are also phosphorylated in PHF-tau from Alzheimer brain. A combination of casein kinase 1delta and glycogen synthase kinase-3beta activities could account for over three-quarters of the serine/threonine phosphorylation sites identified in PHF-tau, indicating that casein kinase 1delta may have a role, together with glycogen synthase kinase-3beta, in the pathogenesis of Alzheimer disease.