This study describes a method for the extraction and simultaneous measurement of cortisone, cortisol and corticosterone using dexamethasone as an internal standard. Solid-phase extraction of plasma steroids with C18 columns allows the samples to be extracted, washed and concentrated in a single step with minimal sample handling and without the use of large volumes of organic solvents. HPLC separation of the steroids is accomplished within 10 min and the individual steroid peaks are quantitated by UV detection at 239 nm. This assay was examined for linearity, extraction efficiency, precision and potential interference by commonly used drugs. Plasma values of glucocorticoids are reported for samples obtained from human subjects as well as from rats. HPLC was also compared to RIA for the determination of plasma levels of corticosterone in the rat. Solid-phase extraction and assay by HPLC provides a rapid and specific method for the simultaneous determination of plasma glucocorticoids.