Morphine induces cardioprotection against ischaemia-reperfusion injury. While aiming to investigate the underlying signal transduction cascade of morphine preconditioning in isolated Langendorff-perfused rat hearts, the expected cardioprotection was not detectable. Thus, we investigated the influence of different preconditioning protocols and substrate conditions on cardioprotection in this experimental model. Isolated rat hearts underwent 35 min global ischaemia followed by 60 min reperfusion. Morphine PC was initiated by 3 cycles of 5 min 1 muM morphine with either 5 min washout [3PC5 (5)] or 15 min washout [3PC5 (15)] before ischaemia; by 15 min morphine with 15 min washout before ischaemia [PC15 (15)]; or by 15 min 10 muM morphine with 15 min washout [PC15 (15)-10 microM]. Ischaemic preconditioning was initiated by 3 cycles of 3 min ischaemia; in another group, hearts received 1 muM morphine continuously for 10 min before ischaemia until the end of reperfusion [continued morphine]. To investigate the effects of glutamine, two groups received a glutamine-free perfusate: a control group, and a morphine preconditioning group [3PC5 (15)]. Ischaemic preconditioning reduced infarct size by 75%, and continued morphine by 46% compared to control group. With the glutamine containing perfusate, none of the morphine PC pretreatments had an effect on infarct size. In glutamine-free perfusate, 3 cycles of 5 min 1 microM morphine with 15 min washout reduced infarct size from 45%+/-8% (control) to 20%+/-5% (3PC5 (15). Cardioprotection by morphine-induced preconditioning is model dependent: in the isolated rat heart, morphine preconditioning is prevented by a glutamine containing perfusate.