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A competitive protein-binding method for the measurement of progesterone in plasma of human subjects was investigated. The purification steps necessary to achieve good accuracy, precision and specificity were determined. It was found that one paper chromatographic separation of unwashed ethyl acetate plasma extracts was sufficient, providing that the sample contains a minimum of 1 ng. progesterone. Water blank values equivalent to 0·05 ng. progesterone were consistently obtained. The concentrations of progesterone found in plasma during the follicular and luteal phases of the menstrual cycle and in male plasma were 0·14 ± 0·14, 0·82 ± 0·74 and 0·022 ± 0·015 (S.D.) µg./100 ml., respectively.