Nitric oxide (NO) is implicated in triggering and mediating delayed preconditioning. However, the role of NO and endothelial nitric oxide synthase (eNOS) in early ischaemic preconditioning is controversial. To investigate the role of eNOS in this paradigm further, the response of hearts isolated from either eNOS knockout (KO) or eNOS wild type (WT) mice to various ischaemic-preconditioning stimuli were studied. To determine whether eNOS is pivotal to early ischaemic preconditioning, hearts from both groups were subjected to a robust ischaemic-preconditioning stimulus consisting of four cycles of 5 min ischaemia and reperfusion (4PC) prior to an injurious ischaemia/reperfusion insult. Both WT and KO animals demonstrated significant attenuation of infarct size from 34.91+/-2.14 to 21.32+/-2.53% and from 30.92+/-1.87 to 22.60+/-2.98%, respectively. Whilst eNOS/NO appears not to be pivotal to early ischaemic preconditioning, NO can nonetheless mediate protection in both WT and KO hearts, as the NO donor, S-nitroso N-acetyl penicillamine (2 microM), was seen to mimic preconditioning protection. Therefore, it appears that the targets for NO mediated protection exist in both WT and KO hearts. Reducing the number of preconditioning cycles from four to three to two continued to result in significant attenuation of infarct size in WT hearts (26.30+/-2.86 and 26.70+/-1.95%). However, early ischaemic preconditioning failed in the KO hearts with any preconditioning stimulus less robust than the four cycle protocol used in the initial experiments (3PC 32.51+/-3.04%, 2PC 28.64+/-2.55%). Therefore, whilst eNOS is not essential for robust early ischaemic preconditioning, NO can be shown to be cardioprotective in its own right, and, moreover, eNOS and thus nitric oxide synthesis may contribute to early ischaemic preconditioning by lowering the ischaemic threshold for protection.