Uroporphyrinogen III synthase (hydroxymethylbilane hydro-lyase (cyclizing); EC 4.2.1.75), the fourth enzyme in the heme biosynthetic pathway, was purified to homogeneity from human erythrocytes. For enzyme purification and characterization, a sensitive coupled enzyme assay was used which generated the substrate, hydroxymethylbilane; the oxidized product, uroporphyrin III, was quantitated by high pressure liquid chromatography. Uroporphyrinogen III synthase was initially separated from delta-aminolevulinate dehydratase and hydroxymethylbilane synthase by a preparative anion exchange chromatographic step. Subsequent chromatography on hydroxyapatite, phenyl-Sepharose, and Sephadex G-100 purified the enzyme about 70,000-fold with an 8% yield. Homogeneous enzyme was obtained following a final C4-reversed phase high pressure liquid chromatographic step which removed a single major and several minor protein contaminants from the enzyme. The purified enzyme had a specific activity of over 300,000 units/mg, an isoelectric point of 5.5, and was thermolabile (t1/2 at 60 degrees C approximately 1 min). Molecular weight studies by gel filtration (Mr approximately equal to 30,000) and analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately equal to 29,500) were consistent with the enzyme being a monomer. Using hydroxymethylbilane as substrate, the purified enzyme formed uroporphyrinogen III in the absence of hydroxymethylbilane synthase or other cofactors. The pH optimum was 7.4 and the Km for hydroxymethylbilane was 5-20 microM. The enzyme was activated by Na+, K+, Mg+, and Ca2+ and was inhibited by Cd2+, Cu2+, Hg2+, and Zn2+. Amino acid composition analysis was performed, and the N-terminal sequence, Met-Lys-Val-Leu-Leu-Leu, was determined by microsequencing. The availability of the purified enzyme should permit investigation of its reaction mechanism as well as facilitate biochemical and molecular studies of the genetic defect in congenital erythropoietic porphyria.