To gain a better understanding of the intracellular sites of antigen processing we have looked at the localization of human immunodeficiency virus (HIV)-1 Nef protein by confocal microscopic and biochemical means. We found that ubiquitin (Ub)-Nef fusion proteins were localized to the centrosome in transfected COS-7 cells, and that the colocalization was inhibited by the microtubule-disrupting agent, nocodazole. Interestingly, we found that Ub-Nef trafficking to the centrosome was not dependent upon the metabolic stability of Ub-Nef nor on the inhibition of proteasome activity. We also analyzed the MHC class I antigen processing of a reporter epitope linked to the Ub-Nef fusion proteins and found that Ub-Nef was processed in COS-7 cells. In addition, we show that this processing was inhibited by nocodazole. We suggest that the centrosome may serve as a site of antigen processing in vivo.