The inner medullary collecting duct (IMCD) is the nephron segment with the highest production of endothelin-1 (ET-1) and the greatest expression of ET-1 receptors that function to adjust Na+ and water balance. We have reported that male rats have reduced natriuresis in response to direct intramedullary infusion of ET-1 compared to females. Our aim was to determine whether alterations of ET-1 receptor expression and downstream intracellular Ca²⁺ signaling within the IMCD could account for these sex differences. IMCDs from male and female rats were isolated for radioligand binding or microdissected for intracellular Ca²⁺ ([Ca²⁺]i) measurement by fluorescence imaging of fura 2-AM. IMCD from male and female rats had similar ET_B expression (655±201 vs. 567±39 fmol/mg protein, respectively) while male rats had significantly higher ET_A expression (436±162 vs. 47±29 fmol/mg protein respectively; p<0.05). The [Ca²⁺]i response to ET-1 was significantly greater in IMCDs from male compared to female rats (288±52 vs. 118±32 AUC, nM x 3 min, respectively; p<0.05). In IMCDs from males, the [Ca²⁺]i response to ET-1 was significantly blunted by the ET_A antagonist BQ-123, but not the ET_B antagonist BQ-788 (control: 137±27, BQ-123: 53±11, and BQ-788: 84±25 AUC, nM x 3 min, respectively; p<0.05), consistent with greater ET_A receptor function in males. These data demonstrate a sex difference in ET_A receptor expression that results in differences in ET-1 Ca²⁺ signaling in IMCD. As activation of ET_A receptors is thought to oppose ET_B receptor activation, enhanced ET_A function in males could limit the natriuretic effects of ET_B receptor activation.