Abstract Toluene-treated cells of Escherichia coli represent an in vitro system capable of replicative DNA synthesis. They should offer advantages for investigation of enzymatic mechanisms of DNA repair synthesis secondary to ultraviolet irradiation. The stability of this system allows in vitro irradiation with subsequent measurement of DNA synthesis. Ultraviolet irradiation in vitro causes an inhibition of replicative DNA synthesis in vitro. Moderate levels of such inhibition are due to a decrease in rate of replication rather than an abrupt cessation. Extensive inhibition is followed by the appearance of DNA synthesis that is ATP-dependent and N-ethylmaleimide-sensitive, but independent of DNA polymerase I or II activity or DNA replication. The ultraviolet irradiation-stimulated DNA synthesis appears to be dependent upon DNA polymerase III activity.