A method for the separation of platelets on the basis of their size has been developed using counterflow centrifugation. Platelets were separated, free of plasma proteins and other cells, into seven subpopulations. The smallest-sized platelets, designated as Fraction 1, had a mean platelet volume (MPV) of 3.94 +/- 0.60 micrometer 3 (SD). Each successive fraction had a progressively larger MPV. The MPV for the largest-sized platelets, designated Fraction 7, was 8.19 +/- 0.64 micrometer 3. The MPV for the original platelets prior to fractionation was 6.57 +/- 0.61 micrometer 3. The mean density of Fraction 1 platelets was 1.067 +/- 0.002 g/cm3, while Fraction 7 had a mean density of 1.072 +/- 0.001 g/cm3. Transmission electron microscopy demonstrated that Fraction 1 had 4.3 +/- 0.9 dense bodies per platelet, and Fraction 7 had 12.6 +/- 2.4 dense bodies per platelet. Platelet LDH activity showed that the Fraction 1 platelets had 4.77 +/- 0.92 iu per 10(10) platelets; Fraction 7 platelets had 14.88 +/- 1.23 iu per 10(10) platelets. The LDH activity in the platelets before separation into subpopulations was 9.47 +/- 1.45 iu per 10(10) platelets. Platelet function was measured by ADP-induced aggregation, serotonin uptake, and thrombin-induced release. Progressively more rapid and more complete aggregation was observed as the platelet size increased over the seven fractions. Serotonin uptake was 4.2 times greater in the Fraction 7 platelets than in the Fraction 1 platelets. Quantitative release of serotonin following thrombin stimulation was significantly greater in the larger-sized platelets than in the smaller-sized platelets. The observed differences in platelet aggregation, dense body content, LDH activity, and serotonin uptake and release suggest that large platelets may be functionally more important than smaller platelets.