The cardiotonic activity of milrinone (Win 47203), a potent analogue of amrinone, was demonstrated in isolated guinea pig, cat, rabbit, rat, and hamster atria and papillary muscles. Milrinone, in concentrations of 0.1–300 μg/ml, caused concentration-dependent increases in guinea pig papillary muscle and atrial developed tension with minimal increases in atrial rate. Compared with the in vitro inotropic activity of amrinone, milrinone was approximately 30 times more potent. The inotropic and chronotropic effects of milrinone do not appear to be mediated by the release of endogenous norepinephrine, by the direct stimulation of β-adrenergic or histaminergic receptors, or through the stimulation of prostaglandin synthesis. The inotropic response of the guinea pig papillary muscles to isoproterenol was potentiated when isoproterenol was given at the peak effect of a minimally effective concentration of milrinone or after prolonged incubation with milrinone. No potentiation was observed when isoproterenol was administered at the peak effect of a high concentration of milrinone, which suggests that the positive inotropic action of milrinone may not be solely attributable to cyclic AMP phosphodiesterase inhibition.