Expression of the murine leukaemia virus (MLV) major Gag antigen p65 G a g using the baculovirus expression system leads to efficient assembly and release of virus-like particles (VLP) representative of immature MLV. Expression of p180 G a g - P o l , facilitated normally in mammalian cells by readthrough of the p65 G a g termination codon, also occurs efficiently in insect cells to provide a source of the MLV protease and a pattern of p65 G a g processing similar to that observed in mammalian cells. VLP release from p180 G a g - P o l -expressing cells however remains essentially immature with disproportionate levels of the uncleaved p65 G a g precursor when compared to the intracellular Gag profile. Changing the p65 G a g termination codon altered the level of p65 G a g and p180 G a g - P o l within expressing cells but did not alter the pattern of released VLP, which remained immature. Coexpression of p65 G a g with a fixed readthrough p180 G a g - P o l also led to only immature VLP release despite high intracellular protease levels. Our data suggest a mechanism that preferentially selects uncleaved p65 G a g for the assembly of MLV in this heterologous expression system and implies that, in addition to their relative levels, active sorting of the correct p65 G a g and p180 G a g - P o l ratios may occur in producer cells.