A recombinant vaccinia virus, vDW4, has been constructed in which the L4R gene encoding the VP8 core DNA-binding protein is inducibly regulated by IPTG (isopropyl beta-D-thiogalactopyranoside). This virus produced normal-sized plaques in the presence of IPTG, but tiny plaques in the absence of inducer. Production of infectious progeny virus was reduced by 97% when VP8 synthesis was repressed and immature virions which had defective interactions between the granular viroplasm and the surrounding virion membrane were evident. However, despite the formation of these abnormal immature virions, virus maturation was able to proceed with the production of mature intracellular naked virus and extracellular enveloped virus particles of normal density. These mature particles were produced to approximately 80% of wild-type levels, but were around 100-fold less infectious. Consistent with the formation of mature virions, the repression of VP8 synthesis did not inhibit proteolytic processing of the major core proteins p4a and p4b. These results suggest that VP8 is required for correct association of the viroplasm and the immature virion envelope and that VP8 must be present during virus assembly for the production of fully infectious progeny virus.