DNA repair synthesis can be specifically measured in osmotically opened, confluent cultured human fibroblasts after exposure to DNA damaging agents such that both induction and mediation of DNA repair synthesis can take place in this cell-free system. Alternatively, by utilizing osmotically shocked, log phase cells and altering the DNA precursors, pH and ionic strength, replicative DNA synthesis can be specifically monitored. Autoradiographic studies show that virtually all of the nuclei from the lysates of the confluent, UV-iradiated cells are lightly labeled in the fashion characteristic of DNA repair. By contrast, only a fraction of nuclei is labeled in a population of unperturbed, opened log phase cells and the labeling is heavy and characteristic of replicative synthesis. Furthermore, equilibrium density gradient sedimentation shows that DNA synthesis in lysates of log-phase cells is semiconservative, whereas that with UV-irradiated cells is repair synthesis. This open cell system has been used to study the enzymology of DNA repair. Thus, dideoxythymidine triphosphate, a specific inhibitor of DNA polymerases beta and gamma, does not inhibit either replicative or repair synthesis. By contrast, aphidicolin, a specific inhibitor of DNA polymerase alpha, inhibits DNA repair and replicative synthesis in both intact and permeabilized cells. Finally, phage T4 UV-exonuclease stimulates repair synthesis, but only when phage T4 UV-endonuclease is also added to the UV-irradiated nuclei.