Human T-cell leukemia virus is a T-cell lymphotropic retrovirus associated with T-cell leukemia and tropical spastic paraparesis, whereas bovine leukemia virus is the etiological agent of enzootic bovine lymphosarcoma. Bovine leukemia virus is reported as the animal model of human T-cell leukemia virus. Just as the viruses themselves, the two retroviral proteinases are very closely related. The bovine leukemia virus and human T-cell leukemia virus proteinases are reported as proteins made of 126 and 125 amino acids respectively belonging to the aspartyl proteinase family. By molecular modelling, we show that bovine leukemia virus and human T-cell leukemia virus proteinases made of only 116 and 115 amino acids respectively display three dimensional structures similar to that observed for other retroviral aspartyl proteinases. The models are based on the three dimensional structures of rous sarcoma virus and human immunodeficiency virus. The native bovine leukemia virus proteinase was isolated from virions with a high degree of purity and, in order to develop suitable inhibitors, we checked that bovine leukemia virus proteinase cleaved oligopeptides at the same site as human T-cell leukemia virus proteinase. Solid phase peptide synthesis was used to produce the putative proteolytic enzyme of bovine leukemia virus (116 amino acids). In this study, we show that the folded synthetic bovine leukemia virus proteinase accurately hydrolyzes a decapeptide corresponding to the sequence of the matrix-capsid cleavage site in the gag polyprotein. In addition, the Leu-1 recombinant human T-cell leukemia virus proteinase (115 amino acids) has been expressed in E.coli and its proteolytic activity tested on synthetic substrate fragments.