Endothelium-dependent relaxation of porcine coronary arteries is attenuated under hypoxic conditions. Recent evidence also indicates that pHimay modulate the release of the endothelium-derived relaxing factor. We tested the hypothesis that hypoxia-induced attenuation of endothelium-dependent relaxation is mediated by alterations in pHi. We developed a novel method for loading surface cells, whereby endothelial cell pHicould be measured in situ on the intact porcine coronary artery. Endothelial cells of arterial ring segments were selectively loaded with the fluorescent indicator BCECF-AM. Differential loading of the endothelial cell layer was verified by confocal microscopy. pHiof the endothelial cells in situ and of endothelium-denuded arteries was measured with a Photon Technology International spectrofluorimeter. The functional integrity of the endothelium was assessed by the endothelium-dependent relaxation to substance P in a paired adjacent ring. In the experimental protocol for pHimeasurements, preparations were perfused with a physiological bicarbonate buffer (pH 7.4), stimulated with KCl (29 mmol/L), and then subjected to hypoxia and reoxygenation. The mean basal pHiin endothelial cells on the intact six arteries was 6.92 +/- 0.07. Addition of KCl to the perfusion medium decreased (P=.025) pHito 6.79 +/- 0.07. Subsequent bubbling with N2increased (P=.009) pHito 7.00 +/- 0.06, which was reversed by reoxygenation. In contrast to the in situ endothelium, pHiof the smooth muscle was not significantly altered from its basal value of 7.24 +/- 0.06 (n=5) by either KCl or hypoxia. This differential behavior corroborated the confocal data indicating differential dye loading. These data thus suggest that oxygen-sensitive alterations in pHimay be an important mechanism of signal transduction in endothelial cells. (Circ Res. 1997;80:21-27.)