To investigate changes at the centrosome during the cell cycle, we analyzed the composition of the pericentriolar material from unsynchronized and S-phase-arrested cells by gel electrophoresis and mass spectrometry. We identified HCA66, a protein that localizes to the centrosome from S-phase to mitosis and to the nucleolus throughout interphase. Silencing of HCA66 expression resulted in failure of centrosome duplication and in the formation of monopolar spindles, reminiscent of the phenotype observed after gamma-tubulin silencing. Immunofluorescence microscopy showed that proteins of the gamma-tubulin ring complex were absent from the centrosome in these monopolar spindles. Immunoblotting revealed reduced protein levels of all components of the gamma-tubulin small complex (gamma-tubulin, GCP2, and GCP3) in HCA66-depleted cells. By contrast, the levels of gamma-tubulin ring complex proteins such as GCP4 and GCP-WD/NEDD1 were unaffected. We propose that HCA66 is a novel regulator of gamma-tubulin function that plays a role in stabilizing components of the gamma-tubulin small complex, which is in turn essential for assembling the larger gamma-tubulin ring complex.