Conventional reversed-phase HPLC conditions have been optimized for resolution of a mixture containing prostaglandins PGE(1), PGE(2), PGF(1alpha), and PGF(2alpha). Electroactive derivative-forming reagents, such as p-nitrobenzyloxyamine, 2-bromo-2'-nitroacetophenone, and 2,4-dinitrophenylhydrazine have been evaluated for use as precolumn reagents for forming prostaglandin derivatives. The results indicate that detection limits of 120 pg are achievable with amperometric detection. The utility of the procedures developed is illustrated by the detection of prostaglandins in human urine and plasma.