Porphobilinogen (PBG) deaminase catalyzes the formation of the linear tetrapyrrole, hydroxymethylbilane, from four molecules of PBG. The tetrapyrrole is either converted to uroporphyrinogen III by uroporphyrinogen cosynthetase or nonenzymatically cyclized to uroporphyrinogen I. Methods for the biosynthesis of gram quantities of PBG and for the determination of PBG-deaminase specific activity in various tissues were developed. Using this assay, the PBG-deaminase activities in human erythrocytes, brain, liver and cultured fibroblasts and lymphoblasts as well as various rat tissues were determined as reference values. In addition, the conditions for the use of this assay in the enzymatic diagnosis of heterozygotes for acute intermittent porphyria were established.