To determine possible mechanisms by which NFTs are formed in Alzheimer's disease (AD), we investigated the ability of tissuetransglutaminase (TGase) to convert human recombinant tau proteins into insoluble filamentous structures. TGase derived from guinea pig liver was activated by calcium to catalyze the in vitro cross-linking of the largest soluble recombinant tau isoform (htau40) into insoluble complexes as determined by electrophoresis following incubation in 4 M urea and SDS. The TGase-catalyzed formation of these insoluble complexes occurred within 15 min to 24 h and the decreased migration of the insoluble material correlated with increased calcium concentrations ranging from 2 mM to 50 mM when analyzed electrophoretically. TGase-treated human recombinant tau formed filamentous structures in vitro that were immunoreactive with antibodies to tau and TGase. These structures retained the insoluble characteristics typical of AD PHF/NFTs. Immunolabeling with the TGase antibody revealed that TGase is associated with the filaments formed from human recombinant tau in vitro as well as with PHFs isolated from NFTs from AD brains. These novel findings support an in vitro model for investigating the biophysical changes that occur in converting soluble tau proteins into an insoluble matrix consistent with the insoluble PHFs/NFTs which may contribute to neuronal degeneration and cell death in the AD brain.