The phosphodiesterase inhibitors milrinone and isobutylmethylxanthine (IBMX) inhibited the conversion of [3H]cAMP to [3H]AMP by washed human platelets in concentration-dependent manners (IC50: milrinone, 2.6 x 10(-6) M; IBMX, 4.6 x 10(-6) M). Milrinone and IBMX increased cAMP levels when stimulated by a single concentration (0.3 microM) of iloprost. EC50:milrinone, 5.6 x 10(-5) M; IBMX, 3 x 10(-5) M. Milrinone was a potent inhibitor of platelet thromboxane A2 (TXA2) synthesis when stimulated by median stimulatory doses of collagen (IC50: 3 x 10(-7) M), sodium fluoride (NaF) (a non-specific G protein activator; IC50: 3 x 10(-7) M) and phorbol ester myristate acetate (PMA) (a protein kinase C activator; IC50: 2.2 x 10(-7) M). In contrast, at median stimulatory doses of A23187 and arachidonate there was a marked decrease in the potency of milrinone in inhibiting TXA2 synthesis. Milrinone had a weak inhibitory effect on TXA2 synthesis when elicited by freeze fracturing. In all experiments IBMX was a weaker inhibitor of TXA2 synthesis, although the general pattern of effects was similar to milrinone. Milrinone inhibited both collagen- and adrenaline-stimulated 45Ca2+ uptake by human platelets in dose-dependent manners. Since platelet TXA2 synthesis is dependent on Ca2+, and milrinone inhibited 45Ca2+ uptake, it is concluded that milrinone exerts its inhibitory effect on platelet activity, principally through an action on Ca2+ mobilisation/binding to effector proteins (protein kinase C and/or phospholipase A2).