Initiation of DNA replication has been observed in vitro with a DNA-membrane complex extracted from Bacillus subtilis. Antibiotics known to interfere with various aspects of initiation inhibited DNA synthesis significantly in vitro, whereas a mutant resistant to one inhibitor failed to respond to its presence. The inhibitory effects occurred primarily when the immediate RNA precursors (ribonucleoside triphosphates) were present in the assay solution but not significantly when the precursors were omitted. Complexes extracted from a temperature-sensitive initiation mutant were almost incapable of synthesizing DNA at the restrictive temperature but displayed extensive synthesis at the permissive temperature. A strong indication of semiconservative DNA synthesis was obtained in vitro after density-shift experiments involving incubation of the complex with a heavy-density DNA precursor, followed by neutral and alkaline CsCl density gradient centrifugation. A significant amount of chain elongation or repair (or both) was also observed.