gamma-Aminolevulinic acid (ALA) dehydratase catalyzes the synthesis of porphobilinogen (PBG) from two molecules of ALA. A semimicro method for the colorimetric determination of ALA dehydratase is presented and applied to various tissues. The enzyme activity in adult male rat liver was 2.22 and 1.94 mumol PBG formed/g liver/h for homogenates assayed with or without dithiothreitol, respectively. The assay was linear for at least 2.5 h and for up to 2.5 mg tissue per assay. The Km for ALA was 4.0 X 10(-4)M and the pH optimum was 6.2-6.4. The effects of activators and inhibitors on enzyme activity are discussed.