Measurement of the ultraviolet circular dichroism of apo-(alkaline phosphatase) in urea solutions showed substantial denaturation in 3m-urea. A zinc-deficient mutant alkaline phosphatase behaved similarly. The stability of the enzyme in 6m-urea was followed as a function of its zinc content and was found to be dependent on the first two of the four zinc atoms bound by apoenzyme. Phosphatase activity was mostly dependent on a second pair of zinc atoms. Mn2+, Co2+, Cu2+ or Cd2+ also restored structural stability. Sedimentation-velocity and -equilibrium experiments revealed that dissociation of the dimer accompanied apoenzyme denaturation in urea concentrations of 1m or higher, without treatment with disulphide-reducing agent.