A sensitive and reproducible fluorescence high-performance liquid chromatography is described for the determination of corticoids and 17-oxosteroids in biological fluids. Δ4-3-oxosteroids and 17-oxosteroids are labelled with dansyl hydrazine in HCl-EtOH solution and in trichloroacetic acid-benzene solution, respectively. In the case of corticoids, steroids are extracted with methylene chloride from 0.1 ml of plasma or 1 ml of urine, labelled with dansyl hydrazine, and then chromatographed on a microparticulate silicagel column (Hitachi gel, No.3042, 4 ×250 mm) with fluorophotometric detector at 350 nm (excitation) and 505 nm (emission) using CH₂Cl₂- EtOH-H₂O (985: 15: 5 or 948: 35: 17) as eluent. 17-oxosteroids are extracted after enzymatic hydrolysis (Helicase) or solvolysis, and assayed by the same manner except the eluent. In this case, CH₂Cl₂-EtOH-H₂O (400: 1: 2) is used as eluent. Linearity of the fluorescence intensity with the amount of all steroids are obtained-greater than 0.5ng. The recoveries were between 90 and 106%, and the coefficient of variations were between 3 and 6%. Comparisons with radioimmunoassay gave a correlation coefficient of 0.978 (n=36) and 0.964 (n=81), for serum cortisol and dehydroepiandrosterone sulfate, respectively. A simultaneous determination of various 17-oxosteroids in urine was successfully carried out by the method. Urinary free cortisol could be analyzed without interference of carbamazepine. After administration of metyrapone, 11-deoxycortisol levels in serum could also be measured rapidly by this method. The method is a clinically useful one for the routine analysis of oxosteroids in blood and urine.