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O2 availability modulates transmembrane Ca²⁺ flux via second-messenger pathways in anoxia-tolerant hepatocytes. J. Appl. Physiol. 82(3): 776-783, 1997.—Transmembrane Ca²⁺-flux was studied from single isolated turtle hepatocytes by using a noninvasive Ca²⁺-selective self-referencing microelectrode. Cells in Ca²⁺-reduced culture medium demonstrated a vanadate-and lanthanum-inhibitable Ca²⁺-efflux of 4 × 10⁻¹⁷ mol Ca²⁺ · μm⁻² · s⁻¹ continuously over 170 h. This flux diminished with 50 nM phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, and was reinstated on PKC deactivation with sphingosine. Progressive hypoxia resulted in a reversible suppression of Ca²⁺ efflux to 90% of normoxic controls with an apparent Michaelis constant for oxygen of 145 μM. PKC activation was critical in this suppression, as anaerobic administration of sphingosine caused a Ca²⁺ influx and cell rupture. Hypoxia was also associated with an altered pattern of adenosine-mediated control over Ca²⁺ efflux. Adenosine (100 μM) elevated Ca²⁺ efflux twofold in normoxia, but neither adenosine nor the A1-purinoreceptor antagonist 8-phenyltheophylline altered the observed anaerobic suppression. Aerobic administration of 2-10 mM KCN failed to reproduce the anaerobic suppression; however, in conjunction with 10 mM iodoacetate, complete metabolic blockade caused a Ca²⁺ influx and cell rupture. These observations suggest modulatory control by oxygen over transmembrane Ca²⁺ efflux involving second-messenger systems in the hypoxic transition.