The previously uncharacterized A30L gene of vaccinia virus has orthologs in all vertebrate poxviruses but no recognizable nonpoxvirus homologs or functional motifs. We determined that the A30L gene was regulated by a late promoter and encoded a protein of approximately 9 kDa. Immunoelectron microscopy of infected cells indicated that the A30L protein was associated with viroplasm enclosed by crescent and immature virion membranes. The A30L protein was also present in mature virions and was partially released by treatment with a nonionic detergent and reducing agent, consistent with a location in the matrix between the core and envelope. To determine the role of the A30L protein, we constructed a stringent conditional lethal mutant with an inducible A30L gene. In the absence of inducer, synthesis of viral early and late proteins occurred but the proteolytic processing of certain core proteins was inhibited, suggesting an assembly block. Inhibition of virus maturation was confirmed by electron microscopy. Under nonpermissive conditions, we observed aberrant large, dense, granular masses of viroplasm with clearly defined margins; viral crescent membranes that appeared normal except for their location at a distance from viroplasm; empty immature virions; and an absence of mature virions. The data indicated that the A30L protein is needed for vaccinia virus morphogenesis, specifically the association of the dense viroplasm with viral membranes.