Modulation of epithelial cell proliferation by the dissolved oxygen concentration (Po ) of the growth medium was assessed with primary human foreskin epithelium and a continuous monkey kidney epitheliai cell line (LLC-MK,). Direct measurement of the growth medium Po2 provides the first quantitative evaluation of epithelial cell proliferation as a function of Po>. Sustained proliferation of LLC-MK, cells occurs in serum-free medium equilibrated with a gas phase containing 18% or 30% 0, viv. Mid-logarithmic phase cultures rapidly consume dissolved oxygen; this results in a 60-70 mm Hg decline in Poz and leads to a stable growth medium Po, between 70 and 100 m m Hg, well above anoxic values. In contrast, if culture medium is equilibrated with a gas phase containing 0% or 1 "/o O2 viv to yield a growth medium Po -20-40 mm tig, proliferation of LLC-MK, and primary foreskin epithelial cells is retarded, and LLC-MK, cells use little dissolved oxygen. Gentle, continuous rocking to prevent diffusion gradient formation enhances proliferation slightly at the higher Pox, but neither periodic fluid renewals nor continued rocking stimulates cells retarded by a lowered oxygen concentration to resume proliferation. The data collectively demonstrate that epithelial cell proliferation requires a Poz > 40 m m Hg, and threshold requirements are probably closer to 70 mm Hg. Glycolysis continues at a Pol insufficient for proliferation, but more lactic acid accumulates in actively proliferating cultures than in cultures equilibrated with 0% oxygen. We conclude that epithelial cells in vitro both consume more oxygen and require a higher Po, for continued proliferation, and that the oxygen requirement for epithelial cell proliferation exceeds that o i a Comparable population of fibroblasts for which low oxygen may enhance survival and proliferation