Current methods for determining plasma prekallikrein, one of three zymogens of the contact phase of plasma proteolysis, are laborious and impractical for general use in a clinical laboratory. Therefore, we have developed a simple, reliable assay using commercially available reagents. By use of the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCI (S-2302), a functional assay, performed in a 96-well microplate, was designed to measure prekallikrein in plasma. Measures were taken to destroy the naturally occurring plasma protease inhibitors of kallikrein without affecting the integrity of the plasma prekallikrein, which allowed complete activation of the zymogen to virtually 100% of predicted activity when compared with that of purified kallikrein. Besides permitting full activation, the use of low pH to destroy critical plasma protease inhibitors allowed the conversion of prekallikrein to kallikrein in as many as 44 plasma samples at one time without the tedious individual timing step usually required to activate each sample. An excellent correlation was found (r = 0.92) when this functional microassay was compared with a functional spectrophotometric assay performed in three subject populations: normal individuals, women receiving oral contraceptives (who frequently exhibit high plasma prekallikrein concentrations), and patients with liver disease (who manifest low plasma prekallikrein levels). This plasma prekallikrein microassay should facilitate the increased determination of plasma prekallikrein in pathophysiologic conditions as well as the monitoring of the progression of various diseases in which contact activation occurs.