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A simple, rapid, sensitive procedure based on solid-phase disk extraction (SPDE) is described for the isolation and concentration of trace levels of selected organochlorine pesticides from human body fluids (serum, cord blood, milk, follicular and seminal fluid). Similar methodology can be used for each matrix; the only restricting factor is the viscosity of the fluid. After denaturizing proteins with formic acid, an Empore™ C18-bonded silica extraction disk cartridge is used for the extraction of the analytes. Subsequent cleanup and lipid removal from the SPDE eluate is achieved by adsorption chromatography on acidified silica or Florisil, depending on the interest in acid-labile pesticides. By using the SPDE procedure, high-throughput parallel-sample processing can be achieved. Instrumental analysis is done by gas chromatography-mass spectrometry in electron-capture negative ionization mode (GC-MS/ECNI). Recoveries for selected organochlorine pesticides range from 65 to 91% (SD <10%) for serum and from 70 to 102% (SD <14%) for milk. Detection limits between 10 and 100 pg/mL fluid can be obtained. The method was validated through successful participation in several interlaboratory tests and through the routine analysis of human serum with various loadings of organochlorine pesticides.