A growing mass of data strongly suggest that a number of vascular disorders induced by oxygen lack, including hypoxic pulmonary vasoconstriction (HPV), are associated with abnormalities of endothelial function but underlined mechanisms still remain unclear. The goal of this study was to shed light on the possible myoendothelial gap junctions contribution to the mechanisms responsible for HPV, and to clarify the phenomenon of paradoxical HPV inversion to relaxation under selective glycolysis inhibition. Membrane potentials were recorded from endothelial cells (EC) of guinea pig main pulmonary arteries (MPA) and aorta using whole-cell patch-clamp technique in current clamp mode. The vascular tone was measured on isolated rat aorta and MPA using contractile recording technique. All animals were anesthetized with sodium pentobarbital (50 mg/kg ip). Hypoxia (pO₂-10 mmHg) caused rapid hyperpolarization in aorta endothelial cells from -24.2±4.1 to -37.4±2.1 mV (n=6). In contrast to this, EC in MPA were depolarized under hypoxic condition from -43.3± 3.2 to -30.6±2.2 mV (n=5). Selective glycolysis inhibition with 10 μM iodoacetic acid (IAA) in combination with 10 mM sodium pyruvate led to inversion of these electrical responses in endothelial cells in both types of vessels, i.e. hypoxia hyperpolarized EC from MPA from -8.5±1.6 to -30.4±3.6 mV (n=6) and depolarized EC from aorta from -28.1±2.4 to -18.2±3.1 mV (n=6). In contractile recording experiments hypoxia elicited constriction in MPA with peak amplitude 45.5±5.3% (n=11) and aorta relaxation to 64.3±6.6% (n=8) of norepinephrine-induced pretone (300 nM). 10 μM IAA reversed HPV to dilatation with amplitude 67.6±7.1% (n=11) while relaxant hypoxia-induced responses in aorta were without changes and consisted 73.4±3.8% (n=8). Gap junction inhibitor, 18β-glycyrrhetinic acid (18β-GA, 30 μM), significantly decreased HPV amplitude to 7.6±5.8% (n=16) and led to inversion of hypoxia-induced aorta dilatation to constriction with amplitude 18.8±7.6% (n=10). After the treatment with 18β-GA selective glycolysis inhibition lost its paradoxical effect on HPV in MPA and hypoxia-induced dilatation in aorta (8.4±2.8% and 7.1±11.2%, respectively). It is likely that one yet unknown glycolysis-controlled mechanism in endothelial cells appears to be a common step for vascular contractility regulation under hypoxia, and that may underlie paradoxical responses of both pulmonary and systemic arterial smooth muscle to hypoxia. Our data suggest also that HPV may be due of depolarization of endothelial cells, which might be conducted from endothelium to smooth muscle cells via myoendothelial gap junctions.