Abstract There are approximately 5.5 Million individuals in South Africa infected with HIV-1, predominantly subtype C (HIV-1C). The emergence of drug resistance to the current Antiretroviral (ARV) regimes is of great concern, thus development of novel, effective drugs/vaccines is vital. Certain conserved and thus vulnerable epitopes within the viral envelope (Env) involved in coreceptor binding are usually protected from the immune system in peripheral blood by the variable loops. However, in immune-privileged sites the Env of CD4-independent viruses may exist in a pre-triggered state where these coreceptor binding epitopes are exposed. Targeting the conserved sites could effectively neutralize HIV-1. This study aimed to adapt an HIV-1C primary isolate towards CD4- independence in the Cf2Th cell line through serial in vitro passage. Primary viruses from 20 drug-naïve HIV-1 AIDS patients were isolated and genotypically and phenotypically characterized. The highest percentage (30%) of CXCR4-usage amongst primary isolates from HIV-1C (and CD recombinant) infected AIDS patients worldwide was detected. These data may illustrate the increasing frequency of HIV-1C CXCR4- utilizing (X4) viruses with time and may support the theory that env is capable of evolving. The emergence/evolution of HIV-1C X4 viruses may have profound implications for viral pathogenesis, disease progression and future use of CCR5 antagonists as ARVs. Longitudinal follow-up studies on larger cohorts may confirm this finding. The CXCR4-utilizing isolate 05ZAFV03 was successfully adapted and serially passaged 12 times through Cf2Th cells, whilst gradually decreasing amounts of CD4 expressing cells numbers over time. Viral growth was detected with 10% CD4 expressing cells however, 100% CD4-independence was not reached. Proviral DNA from each stage of the adaptation process was sequenced and analyzed for mutations acquired within env. The only amino acid change noted was an E152K mutation within the V1 region at passage 4. Overall, the extent of env diversity appears to be a complex relationship between isolate-specific and cell-type specific factors. Future attempts to obtain and characterize an HIV-1C CD4-independent isolate will provide potential sites for therapeutic intervention by compounds such as small molecule inhibitors and/or neutralizing antibodies against the most globally prevalent HIV-1 subtype.