1 This study was performed to characterize the receptor subtypes involved in the endothelin stimulation of phospholipase D (PLD) in rat cortical astrocytes in primary culture. PLD activity was determined by measuring the formation of [ 2 The agonists endothelin-1 (ET-1), endothelin-3 (ET-3), sarafotoxin 6c (S6c) and IRL 1620 elicited PLD activation in a concentration-dependent manner. The potencies of ET-1, ET-3 and S6c were similar. The maximal eects evoked by the ET B -preferring agonists, ET-3, S6c and IRL 1620, were signi®cantly lower than the maximal response to the non-selective agonist ET-1. 3 The response to 1 nM ET-1 was inhibited by increasing concentrations of the ET A receptor antagonist BQ-123 in a biphasic manner. A high potency component of the inhibition curve (24.2+3.5% of the ET-1 response) was de®ned at low (up to 1 mM) concentrations of BQ-123, yielding an estimated K i value for BQ-123 of 21.3+2.5 nM. In addition, the presence of 1 mM BQ-123 signi®cantly reduced the maximal response to ET-1 but did not change the pD 2 value. 4 Increasing concentrations of the ET B selective antagonist BQ-788 inhibited the S6c response with a K i of 17.8+0.8 nM. BQ-788 also inhibited the eect of ET-1, although, in this case, two components were de®ned, accounting for approximately 50% of the response, and showing K i values of 20.9+5.1 nM and 439+110 nM, respectively. The ET-1 concentration-response curve was shifted to the right by 1 mM BQ-788, also revealing two components. Only one of them, corresponding to 69.8+4.4% of the response, was sensitive to BQ-788 which showed a K i value of 28.8+8.9 nM. 5 Rapid desensitization was achieved by preincubation with ET-1 or S6c. In cells pretreated with S6c neither ET-3 nor S6c activated PLD, but ET-1 still induced approximately 40% of the response shown by non-desensitised cells. This remaining response was insensitive to BQ-788, but fully inhibited by BQ-123. 6 In conclusion, endothelins activate PLD in rat cortical astrocytes acting through both ET A and ET B receptors, and this response desensitizes rapidly in an apparently homologous fashion. The percentage contribution of ET A and ET B receptors to the ET-1 response was found to be approximately 20% and 80%, respectively, when ET B receptors were not blocked, and 30 ± 50% and 50 ± 70%, respectively, when ET B receptors were inhibited or desensitized. These results may be relevant to the study of a possible role of PLD in the proliferative eects shown by endothelins on cultured and reactive astrocytes.