To determine whether distinct subpopulations of platelets exist in individual patients, platelets were incubated with monoclonal antibodies to glycoprotein Ib and the glycoprotein IIb-IIIa complex, and analyzed by flow cytometry. Normal donors (n = 15) had single glycoprotein Ib-positive and glycoprotein IIb-IIIa complex-positive populations of platelets, with no subpopulations. In normal donors there was a direct relationship between platelet size and the number of surface glycoprotein Ib and glycoprotein IIb-IIIa complex antigens per platelet. In patients with Bernard-Soulier syndrome and Glanzmann's thrombasthenia, all platelets were equally negative with regard to the glycoprotein Ib and glycoprotein IIb-IIIa complex phenotype, respectively. In contrast, each of six children with chronic myeloid leukemia (four of whom were Philadelphia chromosome negative and two of whom were Philadelphia chromosome positive) had two phenotypically distinct subpopulations of platelets: one glycoprotein Ib negative, the other glycoprotein Ib positive. In each of these six children with chronic myeloid leukemia, phenotypically distinct subpopulations of glycoprotein IIb-IIia complex-negative and glycoprotein IIb-IIIa complex-positive platelets were also detected. Polyclonal antiplatelet antibody bound to both the glycoprotein Ib-negative and glycoprotein IIb-IIIa complex-negative subpopulations. Age-matched controls (n = 3) and adults with Philadelphia chromosome-positive chronic myeloid leukemia (n = 3) showed single glycoprotein Ib-positive and glycoprotein IIb-IIIa complex-positive populations. In conclusion, flow cytometry detected distinct subpopulations of platelets in children with chronic myeloid leukemia. Flow cytometry is an important new method of identification and investigation of subpopulations of platelets in individual patients.