In animal cells, microtubules are organized by centrosomes, which are 1-2 m diameter organelles. The generation of functional centrosome fragments in-vitro through ultrasonication is presented along with microtubule assembly directed by the patterned centrosome fragments. While centrosome fragments are smaller than the fully constituted centrosomes, their microtubule organization function is retained. The centrosome fragment templates offer greater flexibility and better coverage in both patterning and assembly of microtubules when compared with intact centrosomes. This work provides the rationale and potential for the large-area assembly of microtubules and should expand the application of centrosomes and centrosome components for the creation of microtubule-based nanoscale devices. Copyright © 2009 American Scientific Publishers All rights reserved.