The human immunodeficiency virus type 1 gag gene product Pr55(gag) self-assembles when expressed on its own in a variety of eukaryotic systems. Assembly in T lymphocytes has not previously been studied, nor is it clear whether Pr55(gag) particles can package genomic RNA or if the Gag-Pol polyprotein is required. We have used a series of constructs that express Gag or Gag-Pol proteins with or without the viral protease in transient transfections in COS-1 cells and also expressed stably in CD4(+) T cells to study this. Deletion of the p6 domain at the C terminus of protease-negative pr55(gag) did not abolish particle release, while truncation of the nucleocapsid protein reduced it significantly, particularly in lymphocytes. Gag-Pol polyprotein was released from T cells in the absence of pr55(gag) but did not encapsidate RNA. Pr55(gag) encapsidated human immunodeficiency virus type 1 RNA whether expressed in a protease-positive or protease-negative context. p6 was dispensable for RNA encapsidation. Marked differences in the level of RNA export were noted between the different cell lines.