Recent study has shown that monophosphoryl lipid A-induced delayed preconditioning enhanced preservation with cardioplegia and that the protective effects of monophosphoryl lipid A were related to stimulation of calcitonin gene-related peptide (CGRP) release. The purpose of the present study was to explore whether the elevated release of CGRP induced by monophosphoryl lipid A is secondary to stimulation of CGRP synthesis via the nitric oxide (NO) pathway and to characterize the isoform of CGRP. Sprague-Dawley rats were pretreated with monophosphoryl lipid A 24h before the experiment, and then the left main coronary artery of rat hearts was subjected to 1 h occlusion followed by 3 h reperfusion. Infarct size, plasma creatine kinase activity, the plasma level of CGRP, and the expression of CGRP isoforms (aand b-CGRP) mRNA in lumbar dorsal root ganglia were measured. Pretreatment with monophosphoryl lipid A (500mg/kg, i.p.) significantly reduced infarct size and creatine kinase release. Monophosphoryl lipid A caused a significant increase in the expression of a-CGRP mRNA, but not of b-CGRP mRNA, concomitantly with an increase in plasma concentrations of CGRP, and the increased level of CGRP expression happened before stimulation of CGRP release. The effect of monophosphoryl lipid A was completely abolished by pretreatment with Lnitroarginine methyl ester (L-NAME, 10 mg/kg, i.p.), an inhibitor of NO synthase or capsaicin (50 mg/kg, s.c.), which selectively depletes transmitters in capsaicin-sensitive sensory nerves. The results suggest that the delayed cardioprotection afforded by monophosphoryl lipid A involves the synthesis and release of CGRP via the NO pathway, and that the protection is mainly mediated by the a-CGRP isoform.