Encapsidation of the Moloney murine leukemia virus (MMLV) genome is mediated through a specific interaction between the major viral structural protein, Gag, and an RNA packaging signal, ψ. Many studies have investigated this process in vivo, although the specific examination of the Gag-RNA interaction in this context is difficult due to the variety of other viral functions involved in virion assembly in vivo. The Saccharomyces cerevisiae three-hybrid assay was used to directly examine the interaction between MMLV Gag and ψ. In this system, MMLV RNA regions exhibiting high-affinity Gag binding were mapped. All Gag-binding regions were located 3′ to the viral splice donor sequence of the viral RNA transcript. No single short RNA sequence within ψ supported strong Gag interaction. Instead, an RNA comprised of nearly the entire ψ region was necessary to demonstrate an appreciable Gag interaction in the yeast three-hybrid system. These finding support the notion that two stem-loops (C and D) are not sufficient to form a core MMLV encapsidation signal.