The purpose of this study was to investigate vascular preconditioning of individual microvascular networks. Prior work shows that exposure of downstream arterioles to specific agonists preconditions upstream arterioles so that they exhibit an altered local vasoactive response (remote microvascular preconditioning). We hypothesized that mitochondrial K_ATP channels were involved in stimulation of remote microvascular preconditioning (RMP). Arteriolar diameter (~15µm) was observed ~1000Mm upstream of the remote exposure site in the cheek pouch of anesthetized (70mg/kg, pentobarbital) male hamsters (N=104); all agonists were micropipette applied. RMP was initiated by (separately) pinacidil (PIN), diazoxide (DZ), nitroprusside (SNP), or bradykinin (BK) application to the downstream vessel. Fifteen minutes later, RMP was apparent at the upstream observation site by testing local vasoactive responses to L-arg. PIN, DZ, SNP, and BK each stimulated RMP. To evaluate a specific role for mito-K_ATP channels in this response, 5HD (5-hydroxydecanoate), was applied (2^nd pipette) during the downstream stimulation with agonist. 5HD blocked RMP initiated by PIN, DZ, or SNP suggesting that mito-K_ATP are involved prior to SNP signal transduction. To verify this, we applied LNNA (N-nitro-L-arginine) during DZ or SNP stimulation. RMP was blocked for SNP, but not DZ. Thus, stimulation of the remote microvascular preconditioning response requires mito-K_ATP channel activity following stimulation by NO donors.