Fibroblasts, the majority of non-cardiomyocytes in the heart, are known to release several kinds of substances such as cytokines and hormones that affect cell and tissue functions. We hypothesized that undefined substance (s) derived from cardiac fibroblasts may have the potential to protect against ischemic myocardium. To assess our hypothesis, using rats, we investigated: (1) the effect of cardiac fibroblast-conditioned medium (CM) on the viability of hypoxic cardiomyocytes in vitro, (2) the effect of CM on left ventricular (LV) function in global ischemia-reperfusion in an ex vive model, (3) the mechanism underlying cardioprotection by CM. Seventy-two hours after starting a hypoxic culture, the viability of cardiomyocytes was higher (P < 0.05) in the CM treated group (41.4%) compared to the control (20.5%). In Langendorff's preparation, 30 min after ischemia-reperfusion, LV end-diastolic pressure was lower, and LV developed pressure and -LVdP/dt were higher (P < 0.01 or P < 0.05) in the CM group than in the control, although coronary flow did not differ between the two groups. Pretreatment with a protein kinase C inhibitor or a mitochondrial ATP-sensitive K+ channel blocker attenuated these changes of LV function in the CM group. Such cardioprotection was achieved by a fraction of the CM having a molecular weight (MW) > 50,000, but not by that of the CM with a lower MW. In addition, a specific antibody against hepatocyte growth factor (HGF, MW is 84,000) did not reduce the cardioprotection afforded by CM. There may be an unknown cardioprotective substance other than HGF in rats, which mimics ischemic preconditioning and has MW > 50,000.