The vaccinia H5 protein has been implicated in several steps of virus replication including DNA synthesis, postreplicative gene transcription, and virion morphogenesis. Our recent mapping of mutants in the consolidated Condit-Dales collection identified a temperature-sensitive vaccinia mutant in the H5R gene (Dts57). We demonstrate here that Dts57 has a DNA negative phenotype, strongly suggesting a direct role for H5 in DNA replication. We used a temperature shift protocol to determine the impact of H5 temperature sensitivity on postreplicative gene expression and observed changes in the pattern of postreplicative viral mRNA metabolism consistent with a role of H5 in postreplicative transcription. Finally, using a rifampicin release temperature shift protocol, we show that H5 is involved in multiple steps of virion morphogenesis. These data demonstrate directly that H5 plays roles in DNA replication, transcription and morphogenesis in vivo.