AbstractExpression of tau protein in non‐neuronal cells can result in a redistribution of the microtubule cytoskeleton into thick bundles of tau‐containing microtubules (Lewis et al.: Nature 342:498–505, 1989; Kanai et al.: J Cell Biol 109:1173–1184, 1989). We reconstituted microtubule bundles using purified tubulin and tau in order to study the assembly of these structures. Taxol‐stabilized tubulin polymers were incubated with various concentrations of recombinant human tau and examined by electron microscopy. With increasing concentrations of τ3 (tau isoform containing three microtubule binding domains) or τ4 (isoform containing four microtubule binding domains) the microtubules changed orientation from a random distribution to loosely and tightly packed parallel arrays and then to thick cables. In contrast, τ4L, the tau isoform containing four microtubule binding domains plus a 58‐amino acid insert near the N‐terminus, showed minimal bundling activity. τ4‐induced bundling could be inhibited by the addition of 0.5M NaCl or 0.4 mM estramustine phosphate, conditions which are known to inhibit tau binding to microtubules. A tau construct that contained only the microtubule binding domains plus 19 amino acids to the C‐terminus was fully capable of bundling microtubules. Phosphorylation of τ3 with cAMP‐dependent protein kinase had no effect on its ability to induce microtubule bundling. These results indicate that tau protein is directly capable of bundling microtubules in vitro, and suggests that different tau isoforms differ in their ability to bundle microtubule filaments. © 1992 Wiley‐Liss, Inc.