Introduction: Our aim was to develop a robust protocol for inducing ischaemia-reperfusion injury and preconditioning in the in vivo mouse heart. Methods: Animals were prepared by standard techniques adapted for the mouse. Results: Thirty minutes of ischaemia appeared to strike the most appropriate balance between large infarct volumes with the risk of heart failure and excessively small infarcts. With this duration of ischaemia, a protocol of three sets of 5 min of ischaemia and 5 min of reperfusion was found to precondition, leading to significantly smaller infarct sizes (infarct volume: risk zone volume reduced to 10.1+/-2.7% from 41.39%+/-3.0; P<.05, n=6 and 7, respectively). Late preconditioning also occurred (infarct volume: risk zone volume=57.9+/-10.1% vs. 35.4+/-4.2%, sham compared to late preconditioning, respectively; P=.001, n=7). Discussion: Whilst further refinements may indeed be possible, we feel this article details a valuable, robust protocol for in vivo studies of ischaemia-reperfusion injury and preconditioning in the mouse heart.