RSU-1069 is a highly effective hypoxic cell cytotoxin in KHT sarcomas treated in vivo. However, relative to the hypoxic cells, the oxic cells in the tumor appear more sensitive to the drug than would have been predicted on the basis of results with CHO (AA8-4) cells treated in vitro with the drug under oxic and hypoxic conditions. To examine possible reasons for this difference, suspensions of KHT cells were prepared from tumors growing in vivo, and treated with RSU-1069 in vitro under oxic or hypoxic conditions. The sensitivity of the KHT cells was similar to that of AA8-4 cells, regardless of whether the cells were obtained from untreated tumors or from tumors given 15 Gy in vivo just prior to the preparation of the cell suspension. We observed, however, that the sensitivity of both AA8-4 cells and KHT cells to drug treatment under hypoxic conditions increased with the density of the cells in the treated suspension. This result suggests the possibility that a diffusible toxic product may be released from cells. Such a product could contribute to the toxicity of the drug for oxic cells in tumors in situ.