Centrosomes nucleate microtubules and serve as poles of the mitotic spindle. Centrioles are a core component of centrosomes and duplicate once per cell cycle. We previously identified epsilon-tubulin as a new member of the tubulin superfamily that localizes asymmetrically to the two centrosomes after duplication. We show that recruitment of epsilon-tubulin to the new centrosome can only occur after exit from S phase and that epsilon-tubulin is associated with the sub-distal appendages of mature centrioles. Xenopus laevis epsilon-tubulin was cloned and shown to be similar to human epsilon-tubulin in both sequence and localization. Depletion of epsilon-tubulin from Xenopus egg extracts blocks centriole duplication in S phase and formation of organized centrosome-independent microtubule asters in M phase. We conclude that epsilon-tubulin is a component of the sub-distal appendages of the centriole, explaining its asymmetric localization to old and new centrosomes, and that epsilon-tubulin is required for centriole duplication and organization of the pericentriolar material.