Platelets can be damaged easily or activated during isolation, making them unsuitable for functional studies. The most common technique for isolating platelets involves centrifugation. Although gentler methods have been devised to isolate platelets by density gradient centrifugation or electrophoresis, these techniques either result in a relatively dilute platelet preparation or are time-consuming. A simple, gentle technique for isolating concentrated platelet preparations for experimental or clinical use is reported. Freshly drawn whole blood was spun over a commercially available density gradient medium for 30 minutes. The mononuclear cell layer (which also contains most of the platelets) was collected and nucleated cells were pelleted by centrifugation. The recovery of platelets was about 60%. Contamination with leukocytes was less than 1%, and the platelet concentration was about 130% of blood concentration. Higher concentrations can be obtained if more whole blood is layered onto the Mono-Poly Resolving Medium (MPRM; Flow Laboratories, McLean, VA). About 10% of the platelets expressed the activation marker GMP-140 by flow cytometric analysis. They could be activated by thrombin so that 70% to 90% of the platelets expressed GMP-140. Thus, this technique can rapidly and easily yield a functionally intact platelet preparation. This preparation can be purified again if needed. No specialized skills or equipment are needed. A significant advantage of the method is that platelets can be obtained from thrombocytopenic patients in final concentrations that are high enough to use for platelet function testing.