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The culture of EMT6/Ro multicellular tumor spheroids in a 20-fold range of glucose concentrations and a 4-fold range of oxygen tensions had profound effects on the cell subpopulations which developed in the spheroids. As the spheroids increased in diameter, the rate of accumulation of cells with a G₁-phase DNA content was much greater for those spheroids cultured in the lower oxygen and glucose concentrations. Growth fractions of 600- to 800-µm-diameter spheroids, calculated from either DNA content or [³H]thymidine labeling data, showed a direct correlation with the glucose concentrations in the culture medium. The cells from spheroids cultured in low glucose concentrations also showed a more rapid loss in clonogenicity as a function of increasing spheroid diameter. Lowering the oxygen tension enhanced these effects of reduced glucose concentrations. Selective dissociation of 600- to 800-µm-diameter …