We have measured the clonogenic survival of cells isolated directly from colon cancer surgical specimens and treated with 5-fluorouracil (5-FU). Enzymatically dissssociated cells were incorporated into hybrid spheroids, consisting predominantly of nonproliferating HeLa feeder cells. Aliquots were exposed for 1.5 hr to a range of concentrations of 5-FU. From the decrease in clonogenic survival, as deduced from the frequency of colony formers among hybrid spheroids after chemical treatment, we were able to construct survival curves in 50% of the surgical specimens tested. A striking revelation was the presence of a resistant plateau in the survival curves, reminiscent of the solid tumor response to treatment with 5-FU. This resistance was absent in monolayer cultures. Evidence is presented that this resistance is due to the absence of, or delay in, cell cycle progression of cells residing in hybrid spheroids.