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Assembly of vaccinia virus envelopes and immature vaccinia particles was interrupted in HeLa cells treated with rifampin ( rifampicin ). The primary action of rifampin on vaccinia morphogenesis appeared to occur during the stage of envelope formation. When envelopes and immature particles were already present, maturation could continue, even in the presence of rifampin. It was demonstrated that the trilaminar membranes of irregular contour which accumulate in the presence of rifampin are precursors of virus envelopes. When rifampin was removed under controlled conditions, synchronous transitions were observed as the precursor membranes rapidly converted into uniformly curved envelope units with a 10- to 12-nm coat on the convex surface. These experiments provided an opportunity to examine the sequence of some early events in vaccinia morphogenesis. Initially, nascent envelopes remained in clusters around dense viroplasm. Large numbers of single immature particles appeared within 10 min. Nucleation of immature particles was the first evidence of core differentiation and began within 5 to 10 min. Development of lateral bodies and modeling of the biconcave cores was observed within 30 min, and structurally mature virions were present by 2 hr after the removal of rifampin. High resolution autoradiography showed that viral deoxyribonucleic acid, which labeled with 3 H-thymidine during rifampin treatment, was incorporated by the mature vaccinia which formed after rifampin was removed. Concentration of the viral deoxyribonucleic acid in core material evidently occurred after envelope assembly, probably coincident with nucleoid formation. Cytoplasmic crystalloid bodies accumulated during rifampin treatment; they appeared morphologically identical to vaccinia nucleoids and were heavily labeled by 3 H-thymidine.