Porphobilinogen synthase (PBG synthase) gene from Pyrobaculum calidifontis was cloned and expressed in E. coli. The recombinant enzyme was purified as an octamer and was found by mass spectrometry to have a subunit Mr of 37676.59 (calculated, 37676.3). The enzyme showed high thermal stability and retained almost all of its activity after incubation at 70 °C for 16 h in the presence of β-mercaptoethanol (β-ME) and zinc chloride. However, in the absence of the latter the enzyme was inactivated after 16 h although it regained full activity in the presence of β-ME and zinc chloride. The protein contained 4 mol of tightly bound zinc per octamer. Further, 4 mol of low affinity zinc could be incorporated following incubation with exogenous zinc salts. The enzyme was inactivated by incubation with levulinic acid followed by treatment with sodium borohydride. Tryptic digest of the modified enzyme and mass spectrometric analysis showed that Lys257 was the site of modification, which has previously been shown to be the site for the binding of 5-aminolevulinic acid giving rise to the propionate-half of porphobilinogen. P. calidifontis PBG synthase was inactivated by 5-chlorolevulinic acid and the residue modified was shown to be the central cysteine (Cys127) of the zinc-binding cysteine-triad, comprising Cys125, 127, 135. The present results in conjunction with earlier findings on zinc containing PBG synthases, are discussed which advocate that the catalytic role of zinc in the activation of the 5-aminolevulinic acid molecule forming the acetate-half of PBG is possible.