Porphobilinogen synthase (PBGS) is a metalloenzyme that catalyzes the first common step of tetrapyrrole biosynthesis, the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) to form porphobilinogen. Chemical modification data implicate histidine as a catalytic residue of PBGS from both plants and mammals. Histidine may participate in the abstraction of two non-ionizable protons from each substrate molecule at the active site. Only one histidine is species-invariant among 17 known sequences of PBGS which have high overall sequence similarity. In Escherichia coli PBGS, this histidine is His128. We performed site-directed mutagenesis on His128, replacing it with alanine. The mutant protein H128A is catalytically active. His128 is part of a histidine- and cysteine-rich region of the sequence that is implicated in metal binding. The apparent Kd for Zn(II) binding to H128A is about an order of magnitude higher than for the wild type protein. E. coli PBGS also contains His126 which is conserved through the mammalian, fungal, and some bacterial PBGS. We mutated His126 to alanine, and both His126 and His128 simultaneously to alanine. All mutant proteins are catalytically competent; the Vmax values for H128A (44 units/mg), H126A (75 units/mg), and H126/128A (61 units/mg) were similar to wild type PBGS (50 units/mg) in the presence of saturating concentrations of metal ions. The apparent Kd for Zn(II) of H126A and H126/128A is not appreciably different from wild type. The activity of wild type and mutant proteins are all stimulated by an allosteric Mg(II); the mutant proteins all have a reduced affinity for Mg(II). We observe a pKa of approximately 7.5 in the wild type PBGS kcat/Km pH profile as well as in those of H128A and H126/128A, suggesting that this pKa is not the result of protonation/deprotonation of one of these histidines. H128A and H126/128A have a significantly increased Km value for the substrate ALA. This is consistent with a role for one or both of these histidines as a ligand to the required Zn(II) of E. coli PBGS, which is known to participate in substrate binding. Past chemical modification may have inactivated the PBGS by blocking Zn(II) and ALA binding. In addition, the decreased Km for E. coli PBGS at basic pH allows for the quantitation of active sites at four per octamer.