Platelet-derived microparticles (PDMP) appear to play a major role in the generation of procoagulant activity. In this study, we describe a novel flow cytometric method that allows direct evaluation of the procoagulant activity of PDMP and platelets in the physiological milieu of whole blood. The percent PDMP generated in response to calcium ionophore A23187 and calcium was increased in preterm neonates (67.5+/-3.4%, mean +/- S.E.M., n = 8, p <0.05) and term neonates (67.2+/-2.7%, n = 7, p<0.05) compared with adults (49.5+/-3.4%, n = 13). However, in preterm neonates A23187/calcium-induced binding of factor V/Va to PDMP and platelets (22.8+/-5.6 fluorescence units) was markedly reduced (p <0.05) compared to term neonates (58.2+/-7.2) and adults (50.6+/-6.3). In preterm blood, A23187/calcium-induced binding of factor V/Va to PDMP and platelets returned to adult levels when: a) adult plasma, rather than autologous preterm neonatal plasma, was added; or b) factor V, but not factor VIII, was added to autologous preterm neonatal plasma. In summary: 1) We have developed a flow cytometric method for the direct detection of procoagulant PDMP and platelets in whole blood. 2) Compared to adults and term neonates, PDMP and platelets of preterm neonates bound markedly less factor V/Va (reflecting reduced procoagulant activity), because of a relative lack of factor V in preterm neonates. 3) This procoagulant defect in PDMP and platelets may contribute to the propensity of preterm neonates, but not term neonates, to intraventricular hemorrhage. 4) The percent PDMP does not necessarily reflect the degree of procoagulant activity of PDMP or platelets.