Knowledge of the tertiary structure of the proteinase from human immunodeficiency virus HIV-1 is important to the design of inhibitors that might possess antiviral activity and thus be useful in the treatment of AIDS1. The conserved Asp–Thr/Ser–Gly sequence in retroviral proteinases2 suggests that they exist as dimers similar to the ancestor proposed for the pepsins3–5. Although this has been confirmed by X-ray analyses of Rous sarcoma virus and HIV-1 proteinases6,7, these structures have overall folds that are similar to each other only where they are also similar to the pepsins8. We now report a further X-ray analysis of a recombinant HIV-1 proteinase at 2.7 Å resolution. The polypeptide chain adopts a fold in which the N- and C-terminal strands are organized together in a four-stranded β-sheet. A helix precedes the single C-terminal strand, as in the Rous sarcoma virus proteinase6 and also in a synthetic HIV-1 proteinase, in which the cysteines have been replaced by α-aminobutyric acid9. The structure reported here provides an explanation for the amino acid invariance amongst retroviral proteinases, but differs from that reported earlier7 in some residues that are candidates for substrate interactions at P3, and in the mode of intramolecular cleavage during processing of the polyprotein.