The phenomenon of 'ischemic preconditioning' (IP) has been vigorously investigated during the past 15 years. As our knowledge on the possible protective mechanisms of IP has been increasingly expanded, novel approaches based on preconditioning with pharmacological agents have recently emerged. Two drugs have been used to induce delayed preconditioning against myocardial infarction caused by ischemia/reperfusion. One of the drugs was monophosphoryl lipid A (MLA)--a detoxified derivative of lipopolysaccharide from gram-negative strains; and another drug was RC552--a novel synthetic glycolipid that mimics the chemical structure of MLA. We have shown that pretreatment of adult mice with MLA or RC552 (350 microg/kg) 24 h prior to the global ischemia and reperfusion in the isolated perfused heart attenuated myocardial injury. Infarct size was significantly reduced in MLA or RC552-treated groups as compared with the vehicle-treated group. The delayed cardioprotection was associated with a moderate but significant increase of nitric oxide level in the ischemic myocardium. Treatment with S-methylisothiourea (3 mg/kg), a selective inhibitor of inducible nitric oxide synthase (iNOS) abolished MLA or RC552-induced delayed protection. In addition, neither MLA nor RC552 reduced infarct size in iNOS knockout mice. Our findings suggest that both MLA and RC552 are able to induce delayed myocardial preconditioning via iNOS-dependent pathway.