Abstract The time course of the synthesis of certain structural vaccinia virus proteins in mouse L fibroblasts was determined by pulse-labeling infected cells at intervals throughout the multiplication cycle and determining the degree of labeling of individual proteins of progeny virus after polyacrylamide gel electrophoresis. Overall viral structural protein synthesis reaches a peak in these cells at between 6 and 8 hours after infection, but then continues at a relatively high rate throughout the infection cycle. Different groups of viral structural proteins exhibit different patterns of synthesis. The synthesis of two proteins which are constituents of viral cores commences very early, as does that of a protein which is situated near the surface of the virion. These proteins are also synthesized in the presence of cytosine arabinoside, when viral DNA replication is inhibited, and are thus “early” proteins. The synthesis of the two early core proteins, but not that of the surface protein, appears to be subject to switch-off; however, switch-off is not absolute, and small amounts of these proteins continue to be synthesized throughout the infection cycle. The presence of structural viral proteins in “soluble” cell extracts (supernatant solutions after centrifuging at 20,000 g for 60 minutes) was also examined. The structural viral protein complement of such cell extracts bears only limited resemblance to that of virions. Some structural viral proteins appear to be synthesized in the form of polypeptide chains larger than those actually incorporated into virions; other viral proteins appear to aggregate rapidly after their synshesis. This is true particularly of those structural proteins which are the latest to be synthesized. The implications of these results are discussed.