The Gag polyprotein of human immunodeficiency virus (HIV) (Pr55(Gag)) contains sufficient information to direct particle assembly events when expressed within tissue culture cells. HIV Gag proteins normally form particles t a plasma membrane assembly site, in a manner analogous to that of the type C avian and mammalian leukemia/sarcoma viruses. It has not previously been demonstrated that immature HIV capsid can form without budding through an intact cellular membrane. In this study, a rabbit reticulocyte lysate translation reaction was used to recreate HIV capsid formation in vitro. Production of HIV-1 Pr55(Gag) and of a matrix-deleted Gag construct resulted in the formation of a subset of Gag protein structures with an equilibrium density of 1.15 g/ml. Gel filtration chromatography revealed these Gag protein structures to be larger than 2 x 106 Da, consistent with the formation of large multimers or capsid. These Gag protein structures were protease sensitive in the absence of detergent, indicating that did not a complete lipid envelope. Spherical structures were detected by electron microscopy within the reticulocyte lysate reaction mixtures and appeared and appeared essentially identical to immature HIV capsid or retrovirus-like particles. These results demonstrate that the HIV Gag protein is capable of producing immature capsid in a cell-free reaction and that such capsid lack as complete lipid envelope.