1. The zinc content and metal ion dependence of phospholipase C(phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus have been examined. 2. The native enzyme contained about 2 atoms of tightly bound zinc/molecule. 3. Incubation of the enzyme with EDTA or with o-phenanthroline caused inactivation. The inactivation was accompanied by the removal of one zinc atom from the enzyme and could be fully reversed by the addition of Zn2+ or Co2+ to the enzyme and partly reversed by Mn2+ or Mg2+. 4. Prolonged exposure to o-phenanthroline removed the second zinc atom also and produced an enzyme species which was reactivated by Zn2+ only. Full reactivation was accompanied by the binding of about two zinc atoms to the enzyme. 5. The results are consistent with the view that phospholipase C is a zinc metalloenzyme.