The effects of Zn2+, Hg2+, Cd2+, and Cu2+ on normal and lead-inhibited erythrocytic porphobilinogen synthase (PBG-S) were studied in vitro using human whole blood hemolysate. The results demonstrate that each of the divalent ions tested has a characteristic effect on the pH-activity relationship of PBG-S. The effects for a given ion are concentration- and pH-dependent. For Zn2+ these effects are also time-dependent. The results obtained provide an explanation for the contradictory reports of the action of some of the metals in vitro and indicate that future investigations of the effects of metals on an enzyme such as PBG-S are best performed over a judiciously selected pH range rather than at a single pH value. It is also shown that the metals studied will only have a significant effect on the proposed PBG-S activity ratio test for lead intoxication in instances of gross contamination of blood collection devices. The activation and/or inhibition of PBG-S and associate pH-activity profile changes resulting from interaction with the four metal ions tested were attributed to their respective affinity for the thiol and other groups at the active sites. The occurrence of a relatively specific pH optimum for PBG-S after interaction with each ion investigated remains unexplained.