It is well established that cell proliferation in batch (unfed) hematopoietic cell cultures is greatly inhibited relative to that in cultures with feeding. What is not known, however, is the nature of this inhibition. On the basis of our observations in hematopoietic cultures that cell proliferation ceases when the lactate concentration ([lactate]) exceeds 20 mM (accompanied by a decrease in culture pH), we investigated the effect of lactate accumulation on cell proliferation, metabolism, and differentiation. We differ in our approach from previous efforts in that we have tried to more accurately recreate the manner in which lactate accumulates in culture by employing a daily feeding protocol in which [lactate] and/or pH in the fresh medium was adjusted to match the conditions prior to feeding. We conclude that the decrease in pH associated with lactate accumulation significantly inhibits both cell proliferation and metabolism Although inhibition in cultures with high [lactate] and low pH is similar to that in unfed cultures, pH control in unfed cultures does not alleviate the inhibition, indicating that other inhibitory factors are also present. Thus, pH control is necessary, but not sufficient, to eliminate inhibition of cell growth and metabolism in unfed hematopoietic cell cultures. We also conclude that high [lactate] and low pH have little effect on cell differentiation in fed cultures, although there is evidence to suggest that low pH may play a role in monocyte differentiation in unfed cultures.