Milrinone, a potent positive inotropic and vasodilating agent, has shown promise in the clinical treatment of congestive heart failure, but significant controversy about its mechanism of action exists. To approach these mechanistic problems in a non-innervated, non-diffusion-limited system, the effects of milrinone on cultured embryonic chick ventricular cells were examined. At 37 degrees C in physiologic buffer, milrinone produced a rapid, concentration-dependent increase in amplitude of contraction that was 45% of the maximum increment in contraction produced by elevated extracellular calcium; the EC50 was 8 microM. This peak response was quantitatively similar to the contractile response produced by isobutyl methylxanthine, a potent phosphodiesterase inhibitor. Milrinone inhibited 70% of total phosphodiesterase activity of cultured ventricular cells with an EC50 of 11 microM. Exposure to 1 X 10(-4) M milrinone resulted in rapid increase in cyclic AMP content to levels greater than 100% above control within 4 min. The same concentration also produced a 43% increase in the rate of transsarcolemmal 45Ca uptake. The stimulation of 45Ca uptake rate was similar to the response produced by 1 microM isoproterenol and could be completely abolished by 10 microM verapamil. Thus, in cultured embryonic chick myocardial cells, the positive inotropic effect of milrinone is largely, if not entirely, attributable to phosphodiesterase inhibition, leading to intracellular cyclic AMP accumulation and stimulation of transsarcolemmal calcium influx via the slow calcium channel.