1. We have recently demonstrated that glyceryl trinitrate (GTN) exerts a direct myocardial anti-ischaemic effect in both GTN-tolerant and nontolerant rats. Here we examined if this effect is mediated by GTN-derived nitric oxide (NO) and involves guanosine 3'5' cyclic monophosphate (cyclic GMP) and ATP-sensitive K+ channels (KATP). 2. Rats were treated with 100 mg kg-1 GTN or vehicle s.c. three times a day for 3 days to induce vascular GTN-tolerance or nontolerance. Isolated working hearts obtained from either GTN-tolerant or nontolerant rats were subjected to 10 min coronary occlusion in the presence of 10-7 M GTN or its solvent. 3. GTN improved myocardial function and reduced lactate dehydrogenase (LDH) release during coronary occlusion in both GTN-tolerant and nontolerant hearts. 4. Cardiac NO content significantly increased after GTN administration in both GTN-tolerant and nontolerant hearts as assessed by electron spin resonance. However, cardiac cyclic GMP content measured by radioimmunoassay was not changed by GTN administration. 5. When hearts from both GTN-tolerant and nontolerant rats were subjected to coronary occlusion in the presence of the KATP-blocker glibenclamide (10-7 M), the drug itself did not affect myocardial function and LDH release, however, it abolished the anti-ischaemic effect of GTN. 6. We conclude that GTN opens KATP via a cyclic GMP-independent mechanism, thereby leading to an anti-ischaemic effect in the heart in both GTN-tolerant and nontolerant rats.