Parameters of platelet volume have become widely available with the introduction of automated platelet counters. However, variant sample processing and in vitro platelet activation have prevented standardization of platelet volume analysis. We investigated the influence of anticoagulation, storage, temperature, and the presence or absence of RBC on platelet volume. Mean platelet volume (MPV) and the mode of the distribution were calculated from the platelet volume distribution curve recorded with the impedance method and plotted in 27 classes between 1.2 and 22 fl. The effects of EDTA (.335%), citrate (.38%), citrate (.38%)/glutaraldehyde (.125%) and a cocktail containing citrate, forskolin, indomethacin and theophyllin were determined 10, 30, 60, 120 and 240 min after blood collection. Tests were made at 4, 20° and 37°C in whole blood and platelet-rich plasma (PRP)from 6 healthy subjects. Platelet volume was strongly dependent on the anticoagulant in a time-dependent manner. MPV was lowest with citrate/glutaraldehyde and highest with EDTA. The maximum difference was 30% at 60 min both in whole blood and PRP. However, this was true only at 4 and 20°C. At 37°C, there was a constant rise in MPV using citrate/glutaraldehyde exceeding volume changes seen with the other anticoagulants. Platelet volume was higher in whole blood as compared to PRP. The difference was dependent on the anticoagulant used and the incubation time (1.8 fl for EDTA and 1.35 fl for citrate/glutaraldehyde, at 60 min). To determine the influence of platelet loss due to PRP preparation on this effect, we determined platelet volume in parallel from whole blood, PRP and the platelet population separated from whole blood by a linear Percoll gradient (n=5, recovery 94%). MPV was 7.6 fl in whole blood, 5.4 fl in PRP and 6.8 fl from the gradient (anticoagulant: citrate/glutaraldehyde). Platelet volume parameters highly depend on anticoagulation, incubation time and temperature. For clinical studies we recommend anticoagulation with citrate/glutaraldehyde and measurement within 2 hr at room temperature in whole blood.