Cultured cells often exhibit alterations in energy metabolism (increased glycolytic activity and decreased oxidative metabolism) during adaptation to the culture environment. The role of hypoxia as a mediator of these effects was examined by comparison of metabolism in primary rabbit renal proximal tubule (RPT) cultures maintained in stationary culture dishes (DISH), shaking Erlenmeyer flasks (SHAKE), and DISH cultures transferred back to SHAKE conditions (RESHAKE). Both oxidative metabolism and transport capacity were fully preserved in SHAKE cultures over a 24-h period. In contrast, within 6 h, DISH cultures exhibited a continuous decline in transport-dependent and -independent oxygen consumption, respiratory capacity, and ATP and K+ contents. The loss of oxidative metabolism in DISH cultures was accompanied by stimulation of lactate production, detectable within 1 h after plating. Comparison of metabolic properties of DISH cultures to those of RPT exposed to graded levels of hypoxia suggested that medium oxygen tensions may be as low as 1-3% in DISH cultures. RESHAKE cultures exhibited metabolic properties comparable to those of SHAKE cultures, indicating reversibility of DISH culture metabolism on reoxygenation. We concluded that DISH cultures rapidly become hypoxic as a consequence of static culture conditions. Shaking suspension cultures may provide a more metabolically appropriate model for long-term in vitro studies.