The activity of avian sarcoma leukemia virus (ASLV) protease (PR) prior to its release from the precursor protein was determined by introducing mutations at the cleavage site between PR and the adjacent upstream nucleocapsid (NC) protein. Gag DNA fragments containing these mutations were cloned into expression vectors and introduced into Escherichia coli in which the ASLV proteins were expressed. The dipeptide NC-PR containing these mutations did not undergo autoprocessing when expressed in bacterial cells and the fused proteins were devoid of enzymatic activity. However, when the whole Gag polyprotein containing these mutations was expressed in bacterial cells, other PR cleavage sites in the viral Gag polyprotein underwent normal cleavage, indicating that the release of free PR is not a prerequisite for correct processing of the ASLV Gag precursor.