The effects of heparin, protamine, and the heparin-protamine complex on the abilities of platelets to aggregate in vitro in response to adenosine diphosphate (ADP) and epinephrine were determined. Citrated blood was obtained from normal volunteers and portions were treated with heparin, protamine, and three different ratios of heparin and protamine. The threshold concentrations of ADP and epinephrine required to produce complete platelet aggregation were then determined. Compared with control citrated plasma, the geometric mean of the threshold concentration for ADP in the heparinized sample was decreased twofold, from 1.88 to 0.94 micrometer; and that for epinephrine more than threefold, from 0.5 to 0.14 micrometer. In contrast, the threshold concentration for ADP was increased to 3.68 micrometer in the neutralized and to 2.78 micrometer in the overneutralized samples and that for epinephrine to 1.62 micrometer in the neutralized and 1.82 micrometer in the overneutralized samples. These data indicate that heparin increases the sensitivity of platelets to ADP and epinephrine as determined by platelet aggregation, and protamine added to heparinized blood not only reverses this effect, but decreases platelet sensitivity when it is added in concentration that neutralize heparin. Additional protamine has no further effect, and protamine alone has no effect on platelet aggregation.