Polyclonal antisera raised against fusion proteins containing portions of the vaccinia virus D2L and D3R proteins were prepared. Immunoprecipitation of pulse-labeled infected cell extracts and Western blot analysis demonstrated that genes D2L and D3R encode 16.9- and 27-kDa proteins, respectively. Both are synthesized late during infection and there is no evidence for proteolytic processing of either protein. Western blots of purified virus and subvirion fractions showed that D2L and D3R are virion components, residing in a detergent-insoluble fraction, containing viral core structural proteins. Trypsin sensitivity experiments suggest that each is found in an equivalent position within the virus core. Pulse-chase analysis showed that both proteins exhibit biphasic stability in which an unstable nascent component is replaced by a stable form. This observation suggests that the stable component results from the insertion of D2L and D3R into an immature core structure. The DNA sequence of four ts mutants previously mapped to genes D2L and D3R is reported. Analysis of the ability of each mutant to synthesize and process viral proteins showed that protein synthetic patterns were indistinguishable from wild type, however, three of the four mutants were defective in the processing of the major virion structural precursor, p4a. Unlike the biphasic stability observed in wild-type infected cells, D2L and D3R were totally degraded in cells infected at 40 degrees with any of the four ts mutants. Stability of the D2L and D3R proteins, in cells treated with rifampicin, is unaffected which demonstrates that a block in morphogenesis is not directly responsible for the observed instability of the mutant proteins.