Synchronous populations of HeLa S3 cells suffer synergistic killing during S phase in the presence of 0.5-5 mM hydroxyurea together with 5-10 mM caffeine. Both the rate and the extent of killing are greater than expected for independent action of the two drugs. Only simultaneous treatment is effective. The dependence of the synergistic killing on cell age resembles the age dependence for killing by hydroxyurea alone ( > 3 mM), but not that by high concentrations of caffeine. In addition, rapid killing occurs if caffeine is added to cultures that have been incubated in the presence of hydroxyurea from early G1 and are blocked at the beginning of S, although such cells are killed only slowly on continued incubation in 210 mM hydroxyurea alone. Furthermore, cells that are incubated with the two drugs from early GI begin to undergo synergistic killing at about 12 h after mitotic collection, but they do not commence DNA replication for another 2-3 h if the drugs are removed. It is concluded that cells that have reached a point in the cycle identical with or close to the end of GI are sensitive to the combination whether or not they are able to synthesize DNA, and whether or not they are sensitive to hydroxyurea alone. A tentative model is proposed: hydroxyurea is postulated to kill cells by interacting with sites of replication in DNA, and the synergism is attributed to the extra replication points that caffeine is known to induce.