Although stimulated [ 3H] inositol phosphate turnover has been demonstrated in isolated, perfused [ 3H] inositol prelabelled rat hearts, there is still no information regarding Ins(1,4,5)P 3 levels in intact cardiac muscle. Using a D-myo-Ins(1,4,5)P 3 assay system, Ins(1,4,5)P 3 levels were determined in isolated perfused rats hearts during ischaemia, reperfusion and α 1-adrenergic stimulation via noradrenaline (3 × 10 −5 m). Control hearts contained ± 674 pmols Ins(1,4,5)P 3/g dry heart weight. Myocardial Ins(1,4,5)P 3 levels were significantly decreased (± 389 pmols/g dry heart weight) after exposure to 20 mins of normothermic ischaemic cardiac arrest (NICA). Reperfusion produced a marked increase in Ins(1,4,5,)P 3 levels (± 1 115 pmols/g dry heart weight) after only 30 s. Noradrenaline caused a 3–4 fold increase in tissue Ins(1,4,5)P 3 levels within 30 s. After 20 mins stimulation with noradrenaline, the Ins(1,4,5)P 3 levels were still significantly elevated. The rise in tissue Ins(1,4,5)P 3 levels during reperfusion as well as during noradrenaline administration was counteracted by neomycin (0.5 × 10 −3 m), an inhibitor of phosphoinositidase specific phospholipase C. In both events neomycin restored the Ins(1,4,5)P 3 levels to control values. For correlation of tissue Ins(1,4,5)P 3 levels with mechanical events, noradrenaline (3 × 10 −5 m), in the presence of 10 m m LiCl, 10 −7 m propanolol and 10 −7 m atropine, was administered to isolated perfused rat hearts and the mechanical performance recorded over a period of 20 mins. Noradrenaline caused a significant increase in peak systolic pressure and work performance which was maintained for at least 10 mins, suggesting that the positive inotropic effects of noradrenaline may be provoked by Ins(1,4,5)P 3. Furthermore, the finding that 20 min NICA followed by 30 s reperfusion causes an immediate significant increase in Ins(1,4,5)P 3 content suggests a role for the phosphatidylinositol pathway in the intracellular Ca 2+ overloading, characteristic of ischaemia-reperfusion.