Phosphorylation, cleavage and conformational changes in tau protein all play pivotal roles during Alzheimer's disease (AD). In an effort to determine the chronological sequence of these changes, in this study, using confocal microscopy, we compared phosphorylation at several sites (Ser(199/202/396/404/422)-Thr(205) and the second repeat domain), cleavage of tau (D(421)) and the canonical conformational Alz-50 epitope. While all of these posttranslational modifications are found in neurofibrillary tangles (NFTs) at all stages of the disease, we found significantly higher numbers of phospho-tau positive NFTs when compared with cleaved tau (P = 0.006 in Braak III; P = 0.002 in Braak IV; P = 0.012 in Braak V) or compared with the Alz-50 epitope (P < 0.05). Consistent with these findings, in a double transgenic mice model (Tet/GSK-3beta/VLW) overexpressing the enzyme glycogen synthase kinase-3beta (GSK-3beta) and tau with a triple FTDP-17 mutation (VLW) with AD-like neurodegeneration, phosphorylation at sites Ser(199/202)-Thr(205) was greater than truncated tau. Taken together, these data strongly support the notion that the conformational changes and truncation of tau occur after the phosphorylation of tau. We propose two probable pathways for the pathological processing of tau protein during AD, either phosphorylation and cleavage of tau followed by the Alz-50 conformational change or phosphorylation followed by the conformational change and cleavage as the last step.