Media concentration of total soluble CO2 increases with culture age of Tetrahymena pyriformis. CO2 is a weak acid and is capable of acidifying intracellular pH (pHi). Changes in pHi have been demonstrated to affect cell metabolism and growth in many systems. For these reasons, we investigated whether the concentrations of CO2 produced in vitro were sufficient to affect cell proliferation and pHi in Tetrahymena. In this study, we used DMO to mimic the weak acid properties of CO2. DMO is freely permeable to membranes in its uncharged form and has a pKa similar to that of CO2/HCO3(-). In addition, it has the advantages of being metabolically inert and non-volatile. At concentrations similar to endogenously produced CO2, DMO acidifies pHi and arrests culture growth. In addition, procedures are described which decrease the media CO2 concentrations in both growing and non-growing cultures. These conditions lead to increased maximum culture density at stationary phase. The data indicate that, under our conditions, accumulation of CO2 in the culture leads to cessation of growth, probably through elimination of transmembrane pH gradients, which are necessary for regulation of metabolism and growth.