Horse liver alcohol dehydrogenase (EC 18.104.22.168) contains one catalytic and one noncatalytic pair of zinc atoms that can be replaced selectively with cobalt and/or 65zince. We have now prepared "hybird" metalloenzymes by specifically replacing one or both pairs of zinc atoms with 65zinc and/or cobalt. Their differential chemical reactivities serve to characterize the metal atoms at either site.The spectral and kinetic properties of the resultant 65zinc, cobalt, and hybrid enzymes, as well as those of their complexes with 1,10-phenanthroline, identify the metal atoms that are at the catalytic sites and differentiate them from those at the noncatalytic sites. All data are in complete agreement with the results of the x-ray crystal structure analysis. Remarkably, under the conditions used, chemical reactivity, as gauged by thermodynamic methods under equilibrium conditions, identifies the catalytic metal atoms as those which are reactive to 1,10-phenanthroline, while this reagnet does not affect the noncatalytic pair. Under dynamic conditions the kinetics of the metal-metal exchange reveals the converse to be true: the chemical reactivity of the noncatalytic atoms is much higher and, hence, they exchange more rapidly. The results are examined in terms of thermodynamic and kinetic properties of metal complex ions which serve as the basis of possible mechanisms underlying these observations.