Expression of CD9 is a feature of both eosinophils and platelets. We have investigated the CD9 expression on resting and activated eosinophils with regard to possibly interacting platelets. Mixed leukocytes were obtained from the platelet-containing (PC) and platelet-depleted (PD) peripheral blood of healthy donors. A cell membrane permeabilization technique, the FOG method, enabled us to detect the eosinophils as a separate population and permitted flow cytometric analysis of both surface and intracellular antigens. Monoclonal antibodies against CD61 were used to identify platelets. The CD9/CD61 ratio indicated that CD9 on resting eosinophils originates mainly from eosinophils and not from adhered platelets. No difference in CD9 expression was obtained between resting eosinophils in PC and PD blood. However, the expression of CD9 was decreased (p < 0.05) on eosinophils in PMA-activated PD blood but increased (p = 0.001) in PC blood, probably due to interacting platelets since CD61 increased simultaneously. In addition, we were able to detect an intracellularly stored pool of CD9 in eosinophils which decreased after activation with PMA. Together these results indicate a translocation of intracellularly stored CD9 to the cell membrane upon activation, probably followed by a subsequent shedding.