Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, differs from the mammalian C-type retroviruses by preassembling core structures in the cytoplasm of infected cells during morphogenesis. Studies that define the protein composition of M-PMV virions and identify two gag-related polyprotein precursors in M-PMV infected cells are reported. The polyprotein precursor to the internal structural (gag) proteins of M-PMV was identified by immunoprecipitation from lysates of pulse-labeled, virus-infected cells with an antiserum to the major structural protein, p27. Tryptic peptide-mapping experiments have shown that this precursor (Pr78) is cleaved to yield five virion structural polypeptides--p27, pp16, p14, p12, and p10. The pp16 polypeptide represents an additional gag-gene encoded polypeptide, not described previously; it is a phosphoprotein and present in virions in a number of forms. A second gag-related polyprotein precursor, P95, is also present in infected cells although in smaller amounts. This nonglycosylated polypeptide contains all of the leucine-containing tryptic peptides of Pr78 plus three others. Studies of the rate of synthesis and half-life of this protein argue against it being the major gag-gene precursor polypeptide. The possibility that it represents a precursor to the viral protease is discussed.