The current study was based on the uncertainty as to how well monolayer cell cultures growing in customary polystyrene dishes are supplied with O2. For dishes maintained in an air-5% CO2 atmosphere at 37 degrees C, microelectrode measurements revealed that the pericellular steady-state PO2 was 78 mm Hg in confluent bovine endothelial, 110 mm Hg in rat renal mesangial, and 0 (< 0.2) mm Hg in renal (LLC-PK1 and LLC-MK2) or hepatic (HepG2, Hep3B) epithelial cell cultures. These measured PO2 values were in good agreement with those calculated from Fick's law of gas diffusion, applied for the present culture conditions (one-dimensional O2 diffusion, 0.52 cm medium height), the individual cell layer density and the tissue-specific rate of O2 utilization. Our results provide reasons to speculate that conventional monolayer cultures are often hypoxic when incubated in an air-5% CO2 atmosphere. Diffusion-limitations of cellular O2 availability are to be taken into consideration when tissue cultures are used to study PO2-dependent processes.