We have developed a more efficient in vitro replication system for the plasmid R6K with the objective of dissecting the mechanism of activation of replication origins at a distance. Using this in vitro system we have shown that the activation of replication origin gamma of R6K is absolutely dependent on two exogenously added initiator proteins: namely the host-encoded DnaA and the plasmid-encoded Pi proteins. Replication was inhibited by novobiocin, suggesting a requirement for DNA gyrase. Surprisingly, rifampicin stimulated in vitro replication significantly, and this stimulation was manifested in the quantitative enhancement of replication without any noticeable qualitative change in the reaction products. This result suggests that transcription at or near the gamma origin keeps it repressed. Replication intermediates that were allowed to accumulate by dideoxynucleoside triphosphate incorporation were analyzed both by restriction enzyme digestion and by electron microscopy, and both sets of analyses revealed initiation from the gamma origin resulting in theta-type replication intermediates. Further development of this system should help us to understand how DNA-protein interaction at the gamma origin/enhancer activates the distal origins alpha and beta.