A sensitive, specific and precise high-performance liquid chromatographic assay for the simultaneous determination of methylprednisolone, methylprednisone and corticosterone using betamethasone as the internal standard is reported. Rat serum (0.5 ml) is extracted with methylene chloride, washed with sodium hydroxide, then water and the extract is injected onto a microparticulate silica gel column with ultraviolet detection at 254 nm. Calculated limits of quantitation are less than 10 ng/ml and the intra-day coefficient of variation is less than 5% for each steroid. This assay has been applied to preliminary studies of methylprednisolone disposition in the rat. The plasma concentration-time profile for each steroid was determined following intravenous administration of methylprednisolone (10 mg/kg). Peak serum methylprednisone concentrations of ca. 250 ng/ml occurred within 5 min of methylprednisolone administration and the average area under the curve ratio (methylprednisolone/methylprednisone) was 9.3. These findings demonstrate that methylprednisone is a metabolite of methylprednisolone in the rat and suggest that the metabolic back-conversion of methylprednisone to methylprednisolone may be less than in other species.