The human cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) potently restrict human immunodeficiency virus type 1 (HIV-1) replication, but they are neutralized by the viral protein Vif. Vif bridges A3G and A3F with a Cullin 5 (Cul5)-based E3 ubiquitin ligase and mediates their proteasomal degradation. This mechanism has been extensively studied, and several Vif domains have been identified that are critical for A3G and A3F neutralization. Here, we identified two additional domains. Via sequence analysis of more than 2,000 different HIV-1 Vif proteins, we identified two highly conserved amino acid sequences, (81)LGxGxSIEW(89) and (171)EDRWN(175). Within the (81)LGxGxSIEW(89) sequence, residues L81, G82, G84, and, to a lesser extent, I87 and W89 play very critical roles in A3G/A3F neutralization. In particular, residues L81 and G82 determine Vif binding to A3F, residue G84 determines Vif binding to both A3G and A3F, and residues (86)SIEW(89) affect Vif binding to A3F, A3G, and Cul5. Accordingly, this (81)LGxGxSIEW(89) sequence was designated the (81)LGxGxxIxW(89) domain. Within the (171)EDRWN(175) sequence, all residues except N175 are almost equally important for regulation of A3F neutralization, and consistently, they determine Vif binding only to A3F. Accordingly, this domain was designated (171)EDRW(174). The LGxGxxIxW domain is also partially conserved in simian immunodeficiency virus Vif from rhesus macaques (SIVmac239) and has a similar activity. Thus, (81)LGxGxxIxW(89) and (171)EDRW(174) are two novel functional domains that are very critical for Vif function. They could become new targets for inhibition of Vif activity during HIV replication.