1. The inotropic activity of glucagon was compared with catecholamines and cardiac glycosides by in vitro procedures which were able to differentiate between the activities of the latter two groups.2. The frequency-force curve for glucagon resembled that of noradrenaline at low stimulation frequencies (1 and 2/min) and that of ouabain at more rapid frequencies of stimulation.3. Noradrenaline and adrenaline increased the amplitude of contraction of cat papillary muscles and markedly shortened the time to reach peak tension. Ouabain and glucagon increased tension without any change in the time to peak tension.4. Noradrenaline caused a rapid onset and rate of rise of contraction of cat aortic strips, whereas the response to ouabain was slow in onset and rate of development. Glucagon had no effect on this preparation, even at high concentrations.5. Manganese ions caused a shift of the dose-response curve to ouabain and glucagon, but not to noradrenaline or calcium. In 0.5 mM Ca media, the response to ouabain was abolished and the curve to noradrenaline shifted.6. When glucagon was added to an atrial preparation, the time to the initial increase in tension and the time to maximal tension was intermediate between that necessary for noradrenaline and that necessary for cardiac glycosides.7. Propranolol blocked the inotropic response to noradrenaline, but not to either ouabain or glucagon.8. A relative measure of contraction-dependency was described. Cardiac glycosides exhibited a greater degree of contraction-dependency than either noradrenaline or glucagon.9. Adrenaline elevated the depressed plateau of the action potential from calf and sheep Purkinje fibres, but ouabain and glucagon were without effect.10. Electrophysiological measurements demonstrated that moderate concentrations of glucagon exerted only a small effect in prolonging atrial and ventricular action potentials.11. Several pharmacological blocking drugs and other inotropic agents did not potentiate or block the inotropic response to glucagon. Reserpine pretreatment increased the response to glucagon.12. It was concluded that glucagon has its own spectrum of inotropic activity and does not completely mimic the effects of either ouabain or noradrenaline.