Classic ischemic preconditioning confers protection to the vulnerable myocardium following brief periods of ischemia with short intermittent periods of reperfusion. The aims of this study were: (i) to ascertain the protection from preconditioning using a relatively long reperfusion interval; (ii) to see whether this protection exists if preconditioning and long reperfusion is repeated and (iii) to evaluate the effect that an additional preconditioning stimulus has if it is given immediately before the sustained ischemia. Following anesthesia, in-vivo hearts were preconditioned with a 5 minute coronary ligation followed by 10 minutes reperfusion (Group A). This was compared to groups that were preconditioned with 5 minutes ischemia and 1 hour reperfusion (Group B); or 5 minutes ischemia with 1 hour reperfusion, repeated twice (Group C); or 5 minutes ischemia with 1 hour reperfusion repeated twice and followed by 5 minutes ischemia and 10 minutes reperfusion (Group D). Protection was assessed by subjecting each of the above groups to a further 45 minutes of regional ischemia followed by 120 minutes reperfusion. This protocol without prior preconditioning served as a control (Group E). The ratio of the infarcted to risk area was 23.1 +/- 4.1% in group A, 38.3 +/- 3.5% in group B, 58.4 +/- 4.9% in group C, 10.4 +/- 3.1% in group D and 61.8 +/- 6.2% in the control group E. Group D was significantly different from all the other groups. Group B was not different in comparison to the control group E. When a relatively long reperfusion period (Group B) was introduced the preconditioning protection diminished. When this long reperfusion period was repeated (Group C) overall protection was lost. However, when preconditioning was re-introduced alter a long delay (Group D), the protection afforded by it not only returned but appeared to be potentiated.