Aspartic proteases have emerged as targets for substrate-based inhibitor design due to their vital roles in the life cycles of the organisms that cause AIDS, malaria, leukemia, and other infectious diseases. Based on the concept of mimicking the substrate transition-state, we designed and synthesized a novel class of aspartic protease inhibitors containing the hydroxymethylcarbonyl (HMC) isostere. An unnatural amino acid, allophenylnorstatine [Apns; (2 S ,3 S )-3-amino-2-hydroxy-4-phenylbutyric acid], was incorporated at the P1 site in a series of peptidomimetic compounds that mimic the natural substrates of the HIV, HTLV-I, and malarial aspartic proteases. From extensive structure-activity relationship studies, we were able to identify a series of highly potent peptidomimetic inhibitors of HIV protease. One highly potent inhibitor of the malarial aspartic protease (plasmepsin II) was identified. Finally, a promising lead compound against the HTLV-I protease was identified.