While the Vif protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in non-permissive cells, it is rapidly degraded intracellularly. We have previously suggested that the rapid turn-over of Vif is biologically meaningful to prevent detrimental effects of this protein at high expression levels. We now studied the mechanism of Vif degradation by examining the blocking effect of protease inhibitors in pulse/chase experiments and by monitoring the extent of Vif ubiquitination. The rapid turn-over of Vif could be blocked by proteasome inhibitors, and Vif was highly ubiquitinated. Cytoskeletal Vif was found to be more stable than soluble cytosolic Vif. These degradation characteristics of Vif were cell type-independent and observed in both non-permissive and permissive cells. Characterization of a series of vif deletion mutants showed that amino acids predicted to be important for formation of beta-strand structures (amino acid nos. 63-70 and 86-89) were critical for maintaining a normal expression level of Vif and for viral infectivity. Finally, we performed comparative stability analysis of the four HIV-1 accessory proteins. Vif was unique in its short half-life and in the magnitude of the degradation. Taken together, we conclude that the proteasome degradation of HIV-1 Vif is a virologically important process and crucial for the function of Vif.