Downregulation of cell surface CD4 is a characteristic property of all lentiviral Nef proteins. We have used mutational analysis to define regions within HIV-1 Nef that are critical for this biological activity. Two discontinuous regions in Nef, extending approximately from residues 96 to 144 and from residues 175 to 186, are reported to be essential for efficient CD4 downregulation. Interestingly, these sequences coincide with two conserved regions of the Nef protein that are juxtaposed to form a single surface on the known structure of Nef. A third, more amino terminal conserved region in Nef, previously reported to be important for Nef enhancement of virion infectivity, was found to be largely dispensable for CD4 downregulation. These data raise the possibility that Nef may contain two structurally distinct functional domains, only one of which contributes to the CD4 downregulation phenotype.