The activity in vivo of HIV-1 proteinase (PR) was analysed in the baculovirus expression system, using eight different constructs of the prt gene under the control of polyhedrin (PH) promoters of various strengths. None of the active PRs was expressed in substantial quantities, and only PH-fused and/or non-functional PR mutants accumulated in high amounts in insect cells. However, enough PR activity was generated from a lengthened PR construct in insect cells to process Gag polyprotein substrate co-expressed in the same cells in trans. Fusion of the first 58 residues from the PH sequence to the PR N terminus did not significantly change its activity and specificity of cleavage of the Gag substrate. When analysed under mild denaturing conditions, PH-fused or unfused full-length PR point mutants, as well as PH-fused or unfused C-terminal deletion mutants, showed a propensity to multimerize, with a predominant occurrence of dimers. The incorporation of PR into Gag particles was studied using eight Gag-PR fusion constructs, all containing a non-functional PR mutant. The PR domain was fused to the C-terminal p6 domain of Gag (p6gag), or translated in frame with NCp7 (as in frameshifted Gag-Pol polyprotein) and followed by downstream sequences of increasing lengths from the Pol domain or the bacterial beta-galactosidase. The results suggested that the presence of the p6gag domain was detrimental to the encapsidation of polyprotein-embedded PR.