Tissue transglutaminase (EC 2.3.2.13) is a calcium-activated enzyme that cross-links specific substrate proteins into insoluble, protease-resistant, high molecular weight complexes. Because the neurofibrillary tangles in Alzheimer disease have similar biochemical characteristics, and because the microtubule-associated protein tau is the predominant component of these structures, the substrate properties of tau with respect to transglutaminase were investigated. Bovine tau and recombinant human tau isoforms rapidly form high molecular weight, cross-linked polymers on incubation with transglutaminase. Polyamine incorporation assays indicate that bovine tau is an excellent substrate of transglutaminase, with a Km of 10.4 +/- 2.2 microM and a Vmax of 40.9 +/- 4.5 nmol/mg of enzyme/min. Individual recombinant human tau isoforms are not equivalent with respect to transglutaminase, as the smallest isoform T3 (352 amino acids) is not as good a substrate as the larger isoforms T4 (383 amino acids) and T4L (441 amino acids). To determine which segments of the tau protein are susceptible to modification by transglutaminase, tau was labeled with [3H]putrescine by transglutaminase and proteolyzed with alpha-chymotrypsin, and the breakdown products were analyzed. These experiments demonstrate that the enzyme modifies tau at only one or a few discrete sites, primarily in the carboxyl half of the molecule. Thus, the reaction is specific for only a small number of the many glutamine residues in tau. Furthermore, a tau deletion construct (T264) containing a portion of the microtubule-binding domains, which is a substrate of transglutaminase, cannot be cross-linked by the enzyme. This provides evidence that the cross-linking reaction is specific, and requires that the substrates be appropriately associated for cross-linking to occur.