Two noncomplementing vaccinia virus temperature-sensitive mutants, Cts8 and Cts26, were mapped to the A3L gene, which encodes the major virion structural protein, 4b. The two ts mutants display normal patterns of gene expression, DNA replication, telomere resolution, and protein processing during infection. Morphogenesis during mutant infections is normal through formation of immature virions with nucleoids (IVN) but appears to be defective in the transition from IVN to intracellular mature virus (IMV). In mutant infections, aberrant particles that have the appearance of malformed IMV accumulate. The mutant particles are wrapped in Golgi-derived membranes and exported from cells. Purified mutant particles are indistinguishable from wt particles in protein and DNA composition; however, they are defective in a permeabilized-virion-directed transcription reaction despite containing significant (Cts8) or even normal (Cts26) levels of specific transcription enzymes. These results indicate that the 4b protein is required for proper metamorphosis of IMV from IVN and that proper organization of the IMV structure is required to produce a transcriptionally active virion particle.