More than 20.8 million people are infected with HIV in sub-Saharan Africa, with South Africa having one of the fastest growing HIV-1 epidemics, where an estimated 2.4 million people were infected. Thirty-two sera from 25 patients were tested for their ability to neutralize HTLV-III B (IIIB) and four primary isolates representing subtypes B, C, D, and a recombinant gag C/env B type. A CEM-SS cell line-based assay was used and the neutralizing titer was defined as the reciprocal of the highest dilution giving a 50% reduction in p24 antigen production. All isolates were neutralized better by subtype-specific sera, except for the C4714 strain, which was neutralized by both subtype B and C sera. C4714 was neutralized by 18/25 (72%) sera, IIIB by 19/32 (59%) sera, D482 by 7/31(23%) sera, B3245 by 6/29 (21%) sera, and the recombinant B/C1491 isolate by 4/25 (16%) sera. Five sera were unable to neutralize any of the isolates. The V3 region of the isolates used in the neutralization assay was amplified by PCR, directly sequenced, and analyzed to reveal variability between the consensus HIV-1 sequences and the isolates. HIV-1 strain C4714 was neutralized more effectively with the sera tested than the IIIIB laboratory strain. Variability in the amino acid sequence of the V3 region, which can alter the conformation of the V3 loop secondary structure, can influence the neutralization of a particular viral isolate. Vaccine formulations should be broadened to include multiple subtypes, especially C subtypes, which is rapidly spreading worldwide.