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In the Rous sarcoma virus (RSV) Gag protein, the 25 amino acid residues of the p10 domain immediately upstream of the CA domain are essential for spherical immature particle formation. The significance of this finding was not known at the inception of this work. I performed a systematic mutagenesis on this region and found excellent correlation between the amino acid side chains required for in vitro assembly and those that participate in the p10-CA dimer interface in a previously described crystal structure. I then introduced exogenous cysteine residues that were predicted to form disulfide bonds across the dimer interface. Upon oxidation of immature particles a disulfide-linked Gag hexamer was formed, implying that p10 participates in and stabilizes the immature Gag hexamer. This is the first example of a critical interaction between two different Gag domains. Molecular modeling of the RSV immature hexamer performed by a collaborator, Paul Murray, indicates that the N-terminal domains of CA must expand relative to the modeled mature hexamer to accommodate the p10 contact