V3 serotyping is a technique for determining HIV-1 genetic subtype based on the binding of antibodies from patient sera or plasma to synthetic V3 peptides derived from subtype consensus sequences. Variation in the performance of this assay has been attributed to V3 sequence heterogeneity, the degree of which varies with patient disease progression, virus co-receptor usage, and genetic subtype. This study assessed the performance of a competitive peptide enzyme immunoassay (cPEIA) in samples from HIV-1 subtype C infected patients with varying disease profiles, including those with syncytium (SI) and non-syncytium-inducing (NSI) viruses. Out of 90 sera tested, 94.4% reacted strongly against the subtype C peptide. There was no significant difference in assay sensitivity among samples from advanced AIDS patients in which humoral immune response may be lower, nor among SI viruses which carry changes in the V3 sequence. Four samples were found to be cross-reactive with other subtypes and one acutely infected patient sample was non-reactive due to low anti-gp120 antibody titers. A significantly higher number of samples showed secondary reactivity to subtype A, compared to other subtypes (P < 0.005). In conclusion, the assay was able to identify HIV-1 subtype C infection with a high level of sensitivity (94%) irrespective of the stage of disease and therefore provides a valuable resource for the large-scale epidemiological monitoring of the spread of HIV-1 subtypes in South Africa.