AbstractProliferation and differentiation are inversely related in many cell culture systems. The study of inducible systems is facilitated by optimal growth conditions in order that whatever differentiation is observed may be attributed to a specific effect of the inducer, rather than to a nonspecific effect of adverse growth conditions. To investigate the role of CO2supply in an inducible system, the K562 human leukemia cell line inducible for hemoglobin synthesis was studied at 10%, 5% and 1.5% CO2concentrations. The lower the CO2concentration, the higher the percentage of benzidine‐positive cells but the slower the growth rate. This increase in benzidine positivity reflected hemoglobin synthesis as indicated by incorporation of3H‐leucine into globin chains. If, in addition to reducing CO2concentration, the complete medium was replaced by a bicarbonate‐free medium, the percentage of benzidine‐positive cells was further increased and growth further slowed. However, if endogenously produced CO2was retained by sealing the culture vessel, these effects were mitigated. Since addition of ribosides blocked these effects, the mechanism for these effects appears to be inhibition of riboside biosynthesis due to the depletion of CO2as a substrate. The implication of this work is that, for reproducibility in studies of inducible systems in which reduction of proliferation may itself increase the probability of differentiation, the CO2tension, the bicarbonate concentration in the medium and the rate of egress of endogenously produced CO2must be kept c