Purified retrovirus Gag proteins or Gag protein fragments are able to assemble into virus-like particles (VLPs) in vitro in the presence of RNA. We have examined the role of nucleic acid and of the NC domain in assembly of VLPs from a Rous sarcoma virus (RSV) Gag protein and have characterized these VLPs using transmission electron microscopy (TEM), scanning TEM (STEM), and cryoelectron microscopy (cryo-EM). RNAs of diverse sizes, single-stranded DNA oligonucleotides as small as 22 nucleotides, double-stranded DNA, and heparin all promoted efficient assembly. The percentages of nucleic acid by mass, in the VLPs varied from 5 to 8%. The mean mass of VLPs, as determined by STEM, was 6.5 x 10(7) Da for both RNA-containing and DNA oligonucleotide-containing particles, corresponding to a stoichiometry of about 1,200 protein molecules per VLP, slightly lower than the 1,500 Gag molecules estimated previously for infectious RSV. By cryo-EM, the VLPs showed the characteristic morphology of immature retroviruses, with discernible regions of high density corresponding to the two domains of the CA protein. In spherically averaged density distributions, the mean radial distance to the density corresponding to the C-terminal domain of CA was 33 nm, considerably smaller than that of equivalent human immunodeficiency virus type 1 particles. Deletions of the distal portion of NC, including the second Zn-binding motif, had little effect on assembly, but deletions including the charged residues between the two Zn-binding motifs abrogated assembly. Mutation of the cysteine and histidine residues in the first Zn-binding motif to alanine did not affect assembly, but mutation of the basic residues between the two Zn-binding motifs, or of the basic residues in the N-terminal portion of NC, abrogated assembly. Together, these findings establish VLPs as a good model for immature virions and establish a foundation for dissection of the interactions that lead to assembly.