Objective: The mediators of acute exercise-induced preconditioning (EIPC) against ischemia-reperfusion injury are not understood. This study assesses the role of nitric oxide synthase (NOS), a reported mediator of other forms of preconditioning. Methods: Male Fischer 344 rats were divided into five groups (n=6-7): sedentary (SED); exercised 2 days on a treadmill at 20 m/min, 6° grade, for 60 minutes (RUN); sedentary, perfused with 100 µM Nw-Nitro-L-arginine methyl ester hydrochloride (L-NAME) to inhibit NOS (SED/L-N); exercised, perfused with L-NAME (RUN/L-N); exercised in a 4°C environment, perfused with L-NAME (CRUN/L-N). Twenty-four hours following exercise, isolated perfused working hearts were subjected to 22.5 minutes of global ischemia plus 30 minutes of normoxic reperfusion. Left ventricle contents of several putative preconditioning mediators were determined. Results: Post-ischemic recovery of cardiac output times systolic pressure (COxSP) was better in RUN than SED (78.4% vs. 50.2% of pre-ischemia, respectively, P<0.05). Inhibition of NOS did not abrogate the improved recovery in the exercise groups or alter recovery in SED. All exercise groups also displayed improved myocardial efficiency (COxSP/oxygen consumption) post-ischemia and less lactate dehydrogenase (LDH) release (p<0.05). L-NAME appeared to lower LDH release independent of exercise. The only change in antioxidant enzyme activity was a decrease in manganese superoxide dismutase in CRUN/L-N (p<0.05). HSP72 expression increased only in RUN and RUN/L-N and endothelial NOS only in CRUN/L-N (p<0.05). Conclusions: Acute EIPC of the Fischer 344 rat heart is not mediated by NOS and does not require increases in HSP72 or antioxidant enzymes.