Preconditioning (PC) with nitric oxide (NO) donors or agents that increase endothelial nitric oxide synthase (eNOS) activity 24 hrs prior to ischemia/reperfusion (I/R) prevents postischemic leukocyte rolling (LR) and stationary leukocyte adhesion (LA). Since 5'-AMP-activated protein kinase (AMPK) phosphorylates eNOS at Ser1177, resulting in activation, we postulated that AMPK activation may trigger the development of a preconditioned anti-inflammatory phenotype similar to that induced by NO donors. Wild-type (WT) C57BL/6J and eNOS-/- mice were treated with the AMPK agonist 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) 30 min (early AICAR PC) or 24 hrs (late AICAR PC) prior to I/R; LR and LA were quantified in single postcapillary venules in the jejunum using intravital microscopy. I/R induced comparable marked increases in LR and LA in WT and eNOS-/- mice relative to sham (no ischemia) animals. Late AICAR PC prevented postischemic LR and LA while early AICAR PC prevented LA in WT mice. Late AICAR PC was ineffective in preventing I/R-induced LR but not LA in the eNOS-/- mice and the same pattern was seen in wild-type animals treated with the NOS inhibitor L-NAME. Early AICAR PC remained effective in preventing LA in eNOS-/- mice. Our results indicate that both early and late PC with an AMPK agonist produces an anti-inflammatory phenotype in postcapillary venules. Since the protection afforded by late AICAR PC on postischemic LR was prevented by NOS inhibition in WT mice and absent in eNOS-deficient mice, it appears that eNOS triggers this protective effect. In stark contrast, antecedent AMPK activation prevented I/R-induced LA by an eNOS-independent mechanism.