One of the striking features of human immunodeficiency virus, simian immunodeficiency virus (SIV), and other lentiviruses is extensive N glycosylation of the envelope protein. To assess the requirement of each N glycan for viral infectivity, we individually silenced all 23 N glycosylation sites in the gp120 subunit of SIVmac239 envelope protein by mutagenizing the canonical Asn-Xaa-Thr/Ser N glycosylation motif in an infectious molecular clone, attempted to rescue viruses from the clones, and compared the replication capability of the rescued viruses in MT4 cells. The mutation resulted in either the recovery of a fully infectious virus (category I); recovery of a faster-replicating virus, compared with the parental virus (category II); or no virus recovery (category III). These categorically different sites were not distributed randomly but were clustered. The sites of category I were localized largely in the N-terminal half, whereas the sites of categories II and III were localized in the C-terminal region, including the CD4 binding site, and the central part, including the C loop, respectively. To learn how far SIV can tolerate the removal of glycans, multiplex mutagenesis was also attempted. When they were appreciably distant from one another in the primary sequence, up to five sites could be silenced in combination without disturbing infectivity. On the other hand, it was difficult to silence contiguous sites. Thus, it appeared that a certain degree of sugar chain density over the local region had to be preserved. We discuss the potential utility of these variously deglycosylated mutants for clarifying the role of N glycans in SIV replication in vivo, as well as in the host response, and for designing vaccines and the generation of glycoprotein crystals.