The maturation of the human immunodeficiency virus type 1 protease (PR) from the Gag-Pol polyprotein is dependent on the intrinsic proteolytic activity of the dimeric Gag-Pol. Herein, we report the kinetics and conformational stabilities of two unique fusion proteins of the protease. In one, X28-PR, a random sequences of 28 amino acids (X28) was linked to the N terminus of the mature protease. In the second construct, X28-delta TF*PR*delta Pol, X28 is fused to the protease which is flanked at both its termini by short sequences (delta) which correspond to the native sequences of the Gag-Pol precursor. Autoprocessing of the latter protein was prevented by inserting an Ala at the native protease cleavage sites. The measured kinetic parameters and the pH-rate profile of both enzymes are nearly identical to those of the mature protease. However, these fusion proteins are more sensitive to acid and urea denaturation than the mature protease. The decrease in the conformational stability of X28-PR and X28-delta TF*PR*delta Pol is reflected by increases in their apparent dissociation constants (Kd) from < 5 nM to approximately 180 and 25 nM, respectively. These results suggest that subunit interactions and hence the dimer stability of the protease domain in the Gag-Pol polyprotein differ from those of the mature protease. The high Kd of X28-PR further suggests that addition of non-native sequences to the N terminus of the protease destablizes the dimer.