AIMS: To assess a commercial enzyme immunoassay (EIA) for the serotyping of hepatitis C virus (HCV) for routine use in a diagnostic laboratory setting, as well as for noting the serotype prevalence of selected specimens. METHODS: Seventy six serum specimens, submitted to the laboratory for routine hepatitis studies between May 1992 and February 1996 and stored at -20 degrees C, were evaluated. These specimens were categorised into specific hepatic, renal, and paediatric clinical conditions. The specimens all tested positive for HCV antibodies on a screening EIA, with confirmation on a recombinant immunoblot assay (RIBA). Certain specimens were also HCV RNA positive by the reverse transcription polymerase chain reaction (RT-PCR). All the specimens were serotyped using the newly developed serotyping EIA. RESULTS: Twenty seven (35.5%) specimens were typable. Type 5 predominated (56%), followed by type 1 (33%), types 1 and 6 (7%) and type 3 (4%). The serotype 5 specimens showed 85% and 90% reactivity with recombinant antigens c100-3 and c22-3c, respectively; serotype 1 specimens showed 75% and 100% reactivity with these antigens. All serotype 5 specimens reacted with the c33-c antigen, but only 60% of serotype 1 specimens reacted with this antigen. The differences in the reactivity of the serotype 5 and serotype 1 specimens for c33-c antigen in the RIBA were significant, but no significant differences in reactivity for antigens c-1-1, c100-3, and c22-3 were noted. Serotype 3 specimens showed equal reactivity with all four antigens used in the RIBA. CONCLUSION: The serotyping EIA was easy to use, rapid, and cost effective compared with molecular assays. This assay seems to be ideal for the routine diagnostic laboratory setting, but could not be used for certain clinical specimens. The demonstration of serotypes 5, 1, and 3 was not unexpected in this cohort. The occurrence of serotype 6, although concurrent and more likely to be a false cross reaction with serotype 1 peptides, requires confirmation by molecular genotyping before it can be claimed that this type is present in South Africa.