We have developed a simple and rapid method for measuring 11 organophosphorus pesticides (dichlorvos, methidathion, salithion, malathion, fenitrothion, fenthion, parathion, diazinon, ethylthiometon, O-ethyl O-(4-nitrophenyl)phenylphosphonothioate (EPN) and chlorpyrifos) and one metabolite (3-methyl-4-nitrophenol) of fenitrothion in serum and urine of acute poisoning patients by HPLC with a diode-array detector. An aliquot of the biological sample after deproteinization by acetonitrile was injected into a C₁₈ column using acetonitrile-water as a mobile phase. The detection limits in serum and urine ranged from 0.05 to 6.8 μg/ml at a wavelength of 230 nm. This method was successfully applied to two actual cases of acute poisoning.