Subpopulations of malignant cells from primary cultures of human colon carcinoma were characterized with respect to their response to mitomycin (MMC). Growth inhibition assays indicated values of 2.06, 0.93, and 0.33 microM for the concentration of drug giving 50% inhibition of growth for sublines HCT 116b, HCT 116, and HCT 116a, respectively. Alkaline elution of filter-bound DNA from cells exposed to MMC in vitro showed a positive correlation between the amount of DNA cross-linking and growth inhibition as a function of drug concentration. Comparable DNA cross-linking was obtained at MMC concentrations of 10 microM for HCT 116b and 5 microM and HCT 116. The cross-linking of DNA from HCT 116a cells at 5 microM MMC was approximately equal to that from HCT 116 cells at doses between 10 and 20 microM MMC. Cross-link removal as a function of time after drug removal of MMC-treated cells was also measured. There was little difference in the rates of alkaline DNA elution after drug removal between HCT 116b and HCT 116a, suggesting that the ability to repair cross-links was not responsible for the differential sensitivities of the cells to MMC. The relative sensitivities of the subpopulations to MMC were reflected in vivo by MMC treatment of nude BALB/c mice bearing xenografts of the cultured sublines.