We investigated the mechanism of steroid action against activated eosinophils. Blood from allergic patients was mixed with hydrocortisone (HC), and the mixture was incubated and then stimulated with a calcium-ionophore (CaI). The concentration of eosinophil cationic protein (ECP) in the plasma obtained by centrifugation of the blood samples decreased significantly in 1 minute compared with that of the control samples. In order to investigate the mechanism of HC action, we isolated eosinophils from peripheral blood and incubated them with HC for 30 seconds to 15 minutes. Using a monoclonal antibody (EG2) specific for ECP, we studied the distribution of ECP by immunofluorescence and immunoelectron microscopic assays. In the former, the fluorescence intensity of the EG2-stained cells was significantly decreased as early as 30 seconds. In the latter, EG2-positive particles inside specific granules of eosinophils were significantly decreased in number within 1 minute. Accordingly, our data suggest that HC started its action against these specific granules of eosinophils within a very short time. SDS polyaclylamide gel electrophoresis (SDS-PAGE)-based quantitative studies on the amount of ECP in eosinophils revealed a significant decrease in the amount of ECP after HC addition. Therefore, HC would seem to exert its inhibitory action against ECP in activated eosinophils immediately, not via genomic pathways but via its direct action on the specific granules in eosinophils, causing the degeneration or inactivation of ECP itself.