The internal, hypoxic cells of V79 spheroids are preferentially killed by misonidazole, whereas adriamycin is preferentially toxic to the external cells due to diffusion limitations. Evaluation of combination treatments with misonidazole and adriamycin was therefore undertaken in an attempt to optimize and equalize cell killing throughout these spheroids. A fluorescence-activated cell sorter was used to select cells as a function of intracellular adriamycin or Hoechst-33342 fluorescence (and, therefore, as a function of location in the spheroid) for subsequent viability determinations. As expected, preferential toxicity of each agent was easily demonstrable, and with prolonged treatment times with both agents, greater uniformity of killing was achieved throughout the spheroid. Additionally, some suggestion of enhanced adriamycin toxicity to aerobic cells of spheroids chronically exposed to misonidazole was observed, analogous to the increased radiation sensitivity previously reported for similar exposure conditions. These results demonstrate that combinations of chemotherapeutic agents can be chosen to have preferential activity against different subpopulations of cells within V79 spheroids. Presumably, a better understanding of the factors contributing to drug resistance will lead to the application of similar principles for human cancer therapy.