In vivo, T7 DNA replication is initiated 15% of the distance from the genetic left end of the chromosome. This site, the primary origin of replication, consists of a 200-base pair (bp) intergenic segment from 14.5 to 15.0% within which are located two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by a 61-bp AT-rich (79% A + T) region. A fragment of T7 DNA containing the primary origin has been inserted into plasmids in order to facilitate studies on initiation in vitro. Initiation of DNA synthesis can be reconstituted using T7 RNA polymerase, T7 DNA polymerase, and T7 origin-containing plasmid DNAs. DNA synthesis is stimulated greatly by the T7 gene 4 protein, an enzyme that has helicase and primase activities. When T7 gene 4 protein is present, replication primarily yields partially replicated Y-form molecules as observed by electron microscopy. Synthesis is unidirectional and the branches of the Y-form molecules are uniform in size, with the branch point of the Y located at the origin. Using restriction enzyme analysis, DNA synthesis has been shown to proceed in the same direction (rightward with respect to the T7 genetic map) as transcription from the two promoters located at the origin. Initiation of DNA synthesis in the opposite direction requires the addition of a single-stranded DNA-binding protein (Fuller, C.W., and Richardson, C.C. (1985) J. Biol. Chem. 260, 3197-3206). The initial products of DNA synthesis have been analyzed by polyacrylamide gel electrophoresis. These DNAs have 10 to 60 ribonucleotides covalently linked to their 5' termini. These RNA primers arise by transcription from each of the two promoters, phi 1.1A and phi 1.1B, located within the primary origin.