Two methods were used for the preparation of platelet concentrates from fresh and overnight‐stored human ACD blood. In a two‐step method prostoglandin E1was added to platelet rich plasma and the mixture was centrifuged to obtain platelet concentrate. The IBM 2991 Blood Cell Processor was used in a one‐step‐method. The concentrates were stored in the frozen state. Concentrates prepared with the two‐step method had a higher platelet recovery and a lower leukocyte contamination than did concentrates prepared with the one‐step method. Based on serotonin uptake velocity, response to hypotonic stress and available platelet factor 3, no essential differences were observed between platelet concentrates prepared from fresh and overnight‐stored blood. Concentrates prepared with the one‐step method had a higher serotonin uptake velocity than those prepared with the two‐step method. This did not result in a better recovery after