1 The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor-selective agonists (sarafotoxin S6c) and antagonists (BQ-123, BQ-788) and digital image¯uorescence microscopy. 2 Con¯uent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ET A : 70% ET B ), with a density of approximately 3400+280 ET A and 8000+610 ET B receptors/cell (n=3 experiments). The density of ET B , but not ET A receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ET B receptor density such that the ET receptor subtype proportions were approximately equal (55% ET A ; 45% ET B ) and similar to those previously observed in intact rat tracheal smooth muscle. ] i increase was due primarily to the mobilization of IP 3 -sensitive and to a lesser extent ryanodine-sensitive intracellular calcium stores. In contrast, ET B receptor activation was exclusively coupled to extracellular calcium in¯ux. 4 Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ET A receptors at a density of 6100+800 receptors cell 71 (n=3 experiments). Serum deprivation was without eect on either ET receptor subtype proportion or ET A receptor density. Challenge of human airway smooth muscle cells with endothelin-1 provoked a concentrationdependent increase in [Ca 2+ ] i (EC 50 : 15 nM), with a peak [Ca 2+ ] i increase to greater than 700 nM. Furthermore, the ET A -mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores. 5 In summary, rat cultured tracheal airway smooth muscle cells contained both ET A and ET B receptors. ET A receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca 2+ from intracellular stores and a strong rise in [Ca 2+ ] i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ET B receptors increased signi®cantly during cell growth, an eect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ET B receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca 2+ ] i , which appeared to be attributable to the in¯ux of extracellular Ca 2+ . In contrast, the populations of ET receptors and their linkage to [Ca 2+ ] i were markedly dierent in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.