A candidate DNA vaccine pTHgagC expressing the immunodeficiency virus-1 (HIV-1) gag gene from South African isolate Du422 was constructed and characterised. The isolate was selected on the basis of being the closest to the South African subtype C consensus sequence. Sequence analysis of cytotoxic T lymphocyte (CTL) epitopes showed that HIV subtype C-infected individuals have CTL responses to a number of epitopes present in the vaccine, but also revealed a more limited presence of subtype A- and any B-derived epitopes. A high level of expression of the immunogen was demonstrated in human cells and a potent, long-lived CTL response to a single inoculation of the DNA vaccine was elicited in BALB/c mice, which could be significantly increased by a boost vaccination at 4 weeks. This is the first candidate HIV-1 DNA vaccine employing the South African subtype C sequences, and constitutes a part of a vaccine scheduled to enter a clinical evaluation in South Africa in 2004.