Replication of plasmids that depend on the 245-base-pair origin of the Escherichia coli chromosome (oriC) requires many purified proteins that (i) direct initiation to oriC (e.g., dnaA protein), (ii) influence initiations elsewhere (e.g., auxiliary proteins), and (iii) prime and extend DNA chains (e.g., priming and synthesis proteins). For the RNA priming and initiation of new DNA chains, the requirements for both primase and RNA polymerase (RNA pol) [Kaguni, J. M. & Kornberg, A. (1984) Cell 38, 183-190] have been further analyzed. Depending on the levels of auxiliary proteins (topoisomerase I and protein HU), three priming systems can operate: primase alone, RNA pol alone, or both combined. At low levels of auxiliary proteins, primase alone sustains an effective priming system. At higher levels, primase action is blocked, but RNA pol alone can initiate replication, albeit feebly; at these high levels of auxiliary proteins, primase and RNA pol act synergistically. When RNA pol is stalled by an inhibitor or lack of a ribonucleoside triphosphate, primase action is also inhibited. Based on these and other data [van der Ende, A., Baker, T. A., Ogawa, T. & Kornberg, A. (1985) Proc. Natl. Acad. Sci. USA 82, in press], RNA pol can counteract inhibition by auxiliary proteins and thus activate the origin for the priming by primase of the leading strand of the replication fork.