HTLV-I is an oncogenic retrovirus that is associated with adult T-cell leukemia. HTLV-I protease and HTLV-I protease fused to a deca-histidine containing leader peptide (His-protease) have been cloned, expressed, and purified. The refolded proteases were active and exhibited nearly identical enzymatic activities. To begin to characterize the specificity of HTLV-I, we measured protease cleavage of peptide substrates and inhibition by protease inhibitors. HTLV-I protease cleavage of a peptide representing the HTLV-I retroviral processing site P19/24 (APQVLPVMHPHG) yielded Km and kcat values of 470 microM and 0.184 s-1 while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded Km and kcat values of 310 microM and 0.0060 s-1. When the P1' proline of P19/24 was replaced with p-nitro-phenylalanine (Nph), the ability of HTLV-I protease to cleave the substrate (APQVLNphVMHPL) was improved. Inhibition of HTLV-I protease and His-protease by a series of protease inhibitors was also tested. It was found that the Ki values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 microM, while the Ki values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 microM. In comparison, the Ki values for inhibition of pepsin by the pepsin inhibitors ranged from 0.72 to 19.2 nM, and the Ki values for inhibition of HIV-1 protease by the HIV protease inhibitors ranged from 0.24 nM to 1.0 microM. The data suggested that the substrate binding site of HTLV-I protease is different from the substrate binding sites of pepsin and HIV-1 protease, and that currently employed HIV-1 protease inhibitors would not be effective for the treatment of HTLV-I infections.