The primary origin of bacteriophage T7 DNA replication is located 15% of the distance from the left end of the T7 DNA molecule. This intergenic segment is A + T-rich, contains a single gene 4 protein recognition site, and is preceded by two tandem promoters for T7 RNA polymerase [RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6]. Analysis by electron microscopy shows that T7 DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] and gene 4 protein initiate DNA synthesis at randomly located nicks on duplex DNA to produce branched molecules. However, upon the addition of T7 RNA polymerase and ribonucleoside triphosphates 14% of the product molecules have replication bubbles, all of which are located near the primary origin observed in vivo; no such initiation occurs on T7 deletion mutant LG37 DNA, which lacks the primary origin. We have also studied initiation by using plasmids into which fragments of T7 DNA have been inserted. DNA synthesis on these templates is also dependent on the presence of T7 RNA polymerase and ribonucleoside triphosphates. DNA synthesis is specific for plasmids containing the primary origin, provided they are first converted to linear forms.