1. The main objective of the present study was to further evaluate the role of nitric oxide (NO) in delayed cardiac protection against ischaemia-reperfusion injury induced by monophosphoryl lipid A (MLA). 2. For this purpose, rats were administered with either 0.5 or 2.5 mg kg(-1) MLA (i.p.). Eight or 24 h later, in vivo NO production in the heart was analysed by electron paramagnetic resonance (EPR) spin trapping technique. In parallel experiments, hearts were removed and perfused according to Langendorff. Functional ventricular parameters and incidence of ventricular fibrillation (VF) were determined after 30 min global ischaemic insult (37 degrees C) followed by 30 min reperfusion. Vascular reactivity of aortic rings was also assessed. 3. Hearts from rats pretreated with 2.5 mg kg(-1) MLA for 24 h (but not those from rats treated with 0.5 mg kg(-1) MLA for 8 and 24 h, or with 2.5 mg kg(-1) MLA for 8 h) exhibited preservation of ventricular function (LVDP, +/-dP/dtmax) and a reduced incidence of VF (25% vs 87.5% in vehicle control) during reperfusion. At the cardioprotective dose of 2.5 mg kg(-1) (for 8 or 24 h), MLA did not produce alterations of the contractile response of aortic rings to noradrenaline. 4. An increased formation of NO was detected in hearts removed from rats pretreated with 2.5 mg kg(-1) MLA for 8 h, but not in those from rats treated for 24 h (or with 0.5 mg kg(-1) MLA). 5. Pretreatment of the animals with the inhibitors of inducible NO-synthase, aminoguanidine (2x300 mg kg(-1)) or L-N6-(1-Iminoethyl)-lysine (L-NIL, 10 mg kg(-1)) abolished both MLA (2. 5 mg kg(-1))-induced rise of NO production (observed 8 h after MLA) and cardioprotection (observed 24 h after MLA). However MLA-induced cardioprotection was not attenuated when the hearts were perfused with aminoguanidine (150 microM) for 30 min before the ischaemic insult. 6. Altogether, the present data suggest that NO acts as a trigger rather then a direct mediator of the delayed cardioprotective effect of MLA in rat heart.