We describe a specific and precise method for measuring concentrations of cortisol in serum or plasma by liquid chromatography. Cortisol, together with an internal standard, equilenin, is extracted from 1 mL of serum or plasma and analyzed isocratically on a reversed-phase column with a mobile phase of acetonitrile/phosphate buffer (30/70, by vol.), at a flow rate of 2.0 mL/min. The eluted cortisol is detected by its absorption at 254 nm and quantitated by peak height measurements. Each analysis requires no longer than 15 min at the optimum column temperature of 50 degrees C. The lower limit of detection for cortisol is about 2 ng/sample for a standard solution; sensitivity is routinely 5 micrograms/L of serum. Analytical recoveries exceeded 95%, with good day-to-day precision (coefficients of variation between 4 and 7%). Of more than 50 drugs and steroids tested for possible interference, only the steroids cortisone, prednisone, and prednisolone may interfere with the analysis of cortisol.