Mason-Pfizer monkey virus (M-PMV), the prototypical type D retrovirus, assembles immature capsids within the cytoplasm of the cell prior to plasma membrane interaction. Several mutants of M-PMV Gag have been described which display altered transport, assembly, or both. In this report, we describe the use of an in vitro synthesis and assembly system to distinguish between defects in intracellular transport and the process of assembly itself for two previously described gag gene mutants. Matrix domain mutant R55W converts the type D morphogenesis of M-PMV particles into type C and has been hypothesized to alter the transport of Gag, redirecting it to the plasma membrane where assembly subsequently occurs. We show here that R55W can assemble in both the in vitro translation-assembly system and within inclusion bodies in bacteria and thus has retained the capacity to assemble in the cytoplasm. This supports the concept that R55 is located within a domain responsible for the transport of Gag to an intracellular site for assembly. In contrast, deletions within the p12 domain of M-PMV Gag had previously been shown to affect the efficiency of particle formation such that under low-level expression conditions, Gag would fail to assemble. We demonstrate here that the efficiency of assembly in the in vitro system mirrors that seen in cells under expression conditions similar to that of an infection. These results argue that the p12 domain of this D-type retrovirus plays a critical role in the membrane-independent assembly of immature capsids.