Abstract Body: Renal autoregulation is intrinsic to preglomerular microvascular smooth muscle cells (MVSMC) for maintaining stable renal blood flow and glomerular filtration rate during arterial pressure fluctuations. Our recent study suggests that renal ischemia-reperfusion (IR) leads to sex-dependent autoregulatory impairment of afferent arterioles. Renal autoregulation is linked to ATP sensitive-Ca²⁺ signaling in MVSMC via activation of P2X purinoceptors (P2X). ATP vasoconstricts afferent arterioles by increasing intracellular calcium concentration ([Ca²⁺]_i) in male rat MVSMC but female rats have not been studied. We postulate that female MVSMC exhibit highly sensitive ATP-mediated Ca²⁺ responses which may protect females against IR. Blood-perfused juxtamedullary nephron experiments were performed on kidneys of Sprague-Dawley rats 24 hours after IR (60-min, bilateral renal artery clamping). ATP-induced changes in [Ca²⁺]_i were assessed in freshly isolated MVSMC using Fura-2. Superfusion of ATP (10^-8–10^-4 M) elicited concentration-dependent vasoconstriction in sham rats of both sexes. The maximal vasoconstriction was 68±2% of the control diameters at 10^-4 mol/L in males and 72±9% in females (n=3/group). In contrast, ATP-induced vasoconstriction was nearly abolished in both sexes 24 h post-IR. The maximal vasoconstriction only reached 91±6% and 97±3% of control diameter in male and female IR rats (n=3/group, P<0.05 vs. shams), respectively, indicating impaired P2X signaling in afferent arterioles post-IR. ATP exposure increased [Ca²⁺]_i in control MVSMC of both sexes by 0.03±0.00 and 0.10±0.01 in males and 0.19±0.02 and 0.43±0.05 in females at 10^-5 and 10^-4 M ATP, respectively. The ATP-induced increase in [Ca²⁺]_i was 4-7 fold higher in female than in male controls, suggesting that MVSMC from normal female rats exhibit a highly sensitive response to ATP stimulation versus male’s. IR suppressed ATP-induced calcium responses in MVSMC of both sexes. [Ca²⁺]_i increased by 0.02±0.00 and 0.04±0.01 in IR males and 0.09±0.01 and 0.09±0.01 in IR females in response to 10^-5 and 10^-4 M ATP, respectively (P<0.05 vs control, n=60-150 cells/3-4 rats/group). Female IR MVSMC however, still possess higher reactivity to ATP than MVSMC from male shams. In conclusion, our studies suggest that MVSMC of male and female rats handle ATP-Ca²⁺ signaling differently and the stronger ATP-mediated [Ca²⁺]_i response in female MVSMC may protect females against IR-induced kidney injury.