The mouse mammary tumor virus (MMTV) Gag protein directs the assembly in the cytoplasm of immature viral capsids, which subsequently bud from the plasma membranes of infected cells. MMTV Gag localizes to discrete cytoplasmic foci in mouse mammary epithelial cells, consistent with the formation of cytosolic capsids. Unexpectedly, we also observed an accumulation of Gag in the nucleoli of infected cells derived from mammary gland tumors. To detect Gag-interacting proteins that might influence its subcellular localization, a yeast two-hybrid screen was performed. Ribosomal protein L9 (RPL9 or L9), an essential component of the large ribosomal subunit and a putative tumor suppressor, was identified as a Gag binding partner. Overexpression of L9 in cells expressing the MMTV(C3H) provirus resulted in specific, robust accumulation of Gag in nucleoli. Förster resonance energy transfer (FRET) and coimmunoprecipitation analyses demonstrated that Gag and L9 interact within the nucleolus, and the CA domain was the major site of interaction. In addition, the isolated NC domain of Gag localized to the nucleolus, suggesting that it contains a nucleolar localization signal (NoLS). To determine whether L9 plays a role in virus assembly, small interfering RNA (siRNA)-mediated knockdown was performed. Although Gag expression was not reduced with L9 knockdown, virus production was significantly impaired. Thus, our data support the hypothesis that efficient MMTV particle assembly is dependent upon the interaction of Gag and L9 in the nucleoli of infected cells.