Various concentrations of oxygen were used to determine the optimum culture medium PO2 for survival and proliferation of attached human and mouse fibroblasts grown from different inoculum sizes. When T-15 flasks were seeded with less than or equal to 2 X 10(4) cells (less than or equal to 1.3 X 10(3) cells/cm2), the highest plating efficiencies and cell yields were obtained with a culture medium PO2 of 40-60 mm Hg. At higher inoculum sizes (10(5) cells per T-15) used routinely for mass cultured, no difference in cell yield or glycolytic activity was observed between cultures gassed with atmospheric, i.e., 18% O2 (growth medium PO2 approximately equal to 125-135 mm Hg) and those gassed with 1% O2 (growth medium PO2 approximately euqal to 40-60 mm Hg). The enhanced clonal growth observed at the latter PO2 results from an increased proliferation rate rather than more efficient attachment and survival of inoculated cells. Glucose uptake and lactic acid accumulation were increased in sparse cultures sparged with 1% O2. A slight extension of lifespan was observed in WI-38 cells serially subcultured with a gas phase of 1% O2.