The role of the three E. coli DNA polymerases (pol I, II, and III) in the replication of Col E1 DNA and other small plasmids with similar replicative properties was investigated in a soluble in vitro system prepared by freeze-thaw lysis of chloramphenicol-treated cells (Staudenbauer, 1976). Extracts from isogenic mutants of the polA, polB and polC gene loci deficient in pol I, II, and III respectively were examined for their replicative capacity. It was found that polA and polC extracts are deficient in the synthesis of supercoiled plasmid DNA, whereas the polB mutation has not effect. Deficient extracts could be complemented by addition of purified pol I and pol III holoenzyme. Analysis of the in vitro synthesized DNA by alkaline gradient centrifugation indicates that pol I is involved in an early step of the replication cycle whereas pol III is required at a later stage. These conclusions are confirmed by inhibition studies employing arabionsylcytosine triphosphate (aCTP) which is shown to interfere with pol III as well as pol II. The strong inhibitory effect of aCTP on plasmid replication is not influenced by the polB mutation and mimicks the effects of thermal inactivation of polC extracts. It is suggested that aCTP blocks plasmid ENA replication in vitro by interfering with pol III function