A specific in vitro association between adenovirus and pruified rat brain microtubules has been previously demonstrated (R. B. Luftig and R. R. Weihing, 1975). When examined by negative-staining electron microscopy, approximately 90% of the virus associated with microtubules was edge bound, i.e., associated within +/-4 nm of the microtubule edge. Similar results are now found for the association of adenovirus with purified chick brain microtubules. When the content of the high-molecular-weight proteins (MAPs) normally present as projections on the surface of microtubules is depleted by fractionation of cold-depolymerized microtubules on agarose A-15M columns or by brief treatment of polymerized microtubules with trypsin, the percentage of edge-bound microtubule-associated viruses is reduced to a level close to that found for particles such as reovirus, coliphage f2, or polystyrene latex spheres, which randomly associate with microtubules (54 to 64% for column-fractionated microtubules; 45 to 68% for trypsin-treated microtubules). Counts of adenovirus particles specifically bound to microtubules, corrected for variations in microtubule and virus concentrations, gave values 2.5 to 3.5 times higher for unfractionated microtubules than for microtubule-associated protein-depleted microtubules. These results are consistent with the suggestion that the specific association between adenovirus and microtubules is mediated by microtubule-associated proteins.