5-Aminolevulinic acid dehydratase from spinach (Spinacia oleracea), highly purified by immunoprecipitation, was characterized by inhibitor studies, amino acid composition, the mode of substrate binding and electron photomicrography. The results show that the conversion of 5-aminolevulinate to porphobilinogen requires an active arginine residue and the formation of a Schiff base between the enzyme and 5-aminolevulinate. The formation of a Schiff base is well known for bacterial and animal dehydratases. Spinach dehydratase, however, is distinguished by its insensitivity to iodoacetamide, a low content of cysteine residues and a high proportion of acidic amino acids. In addition, electron photomicrographs of spinach dehydratase molecules do not resemble the corresponding images of beef liver dehydratase. The finding that an arginine residue is essential for substrate conversion corroborates the suggestion that the right orientation of the substrate in the active center is dependent on a positive charge.