Titration of matrix-bound ('125)I-labelled subunits with soluble nascent ('131)I-labelled subunits resulted in the recovery of over 60% of the original dimer phosphatase activity and a final isotope ratio of 1.06. Titration of metal-free immobilized dimer and monomer with ('65)Zn('2+) showed that the monomer bound 0.9 equivalents of Zn('2+), while the dimer bound 4.1 equivalents of Zn('2+). These results indicate that although the monomer lacks catalytic activity, it exists as a highly folded structure containing sites for Zn('2+) binding and subunit interactions. Chemical modification with ethoxyformic anhydride has demonstrated that three histidines per subunit are modified in the soluble enzyme with a concomitant loss of catalytic activity. Zn('2+) ions protect the enzyme from modification as well as from inactivation, thus implicating all three histidines in Zn('2+) binding. Zn('2+) also protects the monomer against this modification providing independent evidence for Zn('2+) binding in renatured subunits. These techniques, developed in order to characterize subunits of alkaline phosphatase, are generally applicable to other oligomeric enzymes.