The second helix in the C-terminal domain of retroviral capsid (CA) protein functions as the site of dimerization between subunits in capsid assembly and is believed to participate in a unique interface between Gag molecules in immature particles. This study reports isolation of two substitutions in the dimerization helix of Rous sarcoma virus CA protein that have the ability to suppress lethal defects in core maturation imposed by alterations to the major homology region (MHR) motif just upstream. Together with two previously published suppressors, these define an extended region of the dimerization helix that is unlikely to contribute directly to CA-CA contacts but whose assembly-competence may be strongly affected by conformation. The broad-spectrum suppression and temperature-sensitivity exhibited by some mutants argues that they act through modulation of protein conformation. These findings provide important biological evidence in support of a significant conformational change involving the dimerization helix and the MHR during maturation.