We examined the notion that the expression, distribution and activity of the ET-1 receptors are altered in cells from the pulmonary artery of a sheep model of chronic pulmonary hypertension (CPH) induced by continuous air embolization (CAE). Subendothelial (L1) and inner medial (L2) cells from main pulmonary artery were cultured from control (n=3) and hypertensive sheep (n=3). Expression of the ET_A and ET_B receptor genes was assessed by quantitative real-time RT-PCR (n=3-8). ET-1 receptor distribution was examined by FACS analysis (n=5-6) and by binding of [I¹²⁵]-labeled ET-1 (n=4). Receptor activity was assessed by internalization of exogenous ET-1 (measurement of intracellular ET-1 by ELISA) with and without exposure to 10 nM ET-1 and with and without pretreatment with the ET_A and ET_B receptor antagonists, BQ-610 (0.5 µM) and BQ-788 (25 µM), respectively. Basal gene expression of both receptors was significantly higher in L2 than L1 cells. Hypertensive L2 cells showed significantly higher ET_B gene expression when compared to controls (control, 33.4±5.1 real-time RT-PCR data normalized to G3PDH; hypertensive, 56±7.2); expression of the ET_A gene was unchanged. Expression of ET_A and ET_B genes in hypertensive L1 cells was similar to controls. FACS analysis expressed as percent specific fluorescence (%Fsp) confirmed these findings demonstrating a significant increase in ET_B expression only in hypertensive L2 cells when compared to controls (control, 74.2±2.3; hypertensive, 82±2.62, p<0.05). While only the ET_A receptors showed significant binding in control L2 cells at one hour, both receptors bound [I¹²⁵]-labeled ET-1 to the hypertensive cells. Exposure to exogenous ET-1 for 18 hours revealed that only the L2 cells internalized ET-1. ET-1 internalization by the hypertensive L2 cells was significantly reduced when compared to controls (p <0.05). Treatment with ET_A and ET_B receptor antagonists demonstrated that both receptors contributed to internalization of ET-1 in control L2 cells. Treatment of the hypertensive cells with either antagonist alone failed to inhibit internalization of ET-1 but when used in combination significant suppression of ET-1 internalization was found (p <0.05). We conclude that in sheep receiving CAE, alterations in ET_B receptors in cells of the inner medial layer may contribute to the maintenance of CPH via alterations in their expression, distribution and activity.