We previously reported that inhibition of endosomal/lysosomal function can dramatically enhance human immunodeficiency virus type 1 (HIV-1) infectivity, suggesting that under these conditions productive HIV-1 infection can occur via the endocytic pathway. Here we further examined this effect with bafilomycin A1 (BFLA-1) and show that this enhancement of infectivity extends to all HIV-1 isolates tested regardless of coreceptor usage. However, isolate-specific differences were observed in the magnitude of the effect. This was particularly evident in the case of the weakly infectious HIV-1_SF2, for which we observed the greatest enhancement. Using reciprocal chimeric viruses, we were able to determine that both the disproportionate increase in the infectivity of HIV-1_SF2 in response to BFLA-1 and its weak infectivity in the absence of BFLA-1 mapped to its envelope gene. Further, we found HIV-1_SF2 to have lower fusion activity and to be 12-fold more sensitive to the fusion inhibitor T-20 than HIV-1_NL4-3. Proteasomal inhibitors also enhance HIV-1 infectivity, and we report that the combination of a lysosomal and a proteasomal inhibitor greatly enhanced infectivity of all isolates tested. Again, HIV-1_SF2 was unique in exhibiting a synergistic 400-fold increase in infectivity. We also determined that inhibition of proteasomal function increased the infectivity of HIV-1 pseudotyped with vesicular stomatitis virus G protein. The evidence presented here highlights the important role of the lysosomes/proteasomes in the destruction of infectious HIV-1_SF2 and could have implications for the development of novel antiviral agents that might take advantage of these innate defenses.