HIV Nef protein downregulates the surface expression of CD4 on T-cells but its mechanism of activity is unknown. We have analyzed the relevance of different portions of the CD4 molecule with respect to the activity of Nef. Upon transfection of Jurkat T-cells that express Nef or do not express Nef with constructs that include different domains of the CD4 molecule, we demonstrated that Nef downregulation of CD4 requires the first 30 amino acids of the CD4 cytoplasmic tail and that the lck-binding domain of CD4 was at least partially responsible for this effect. Furthermore, upon transfection of U-937 cells with an lck-expressing plasmid we showed that CD4 downregulation by Nef is significantly more efficient when lck is present. Finally, by measuring the rate of CD4 endocytosis by a novel flow cytometric method we showed that the effect of Nef on CD4 surface expression resulted from accelerated CD4 internalization.