Abstract The binding of 2,2-dipyridyl to metal substituted horse liver alcohol dehydrogenase was measured by spectrophotometric titrations. Large changes in the visible absorption spectra were seen for the Co 2+, Cu 2+ and Ni 2+ hybrids upon coordination of 2,2-dipyridyl, due to a change in coordination number. The formation constants for binding to the Co 2+ and Cd 2+ hybrids are of the order 10 6 M −1, which means that these hybrids have a 500-fold higher affinity for 2,2-dipyridyl than the native Zn 2+ enzyme. 2,2-dipyridyl has a 100-fold higher affinity for enzyme bound Cd 2+ than for aqueous Cd 2+ ions, while for Cu 2+ and Zn 2+ the opposite is the case. None of the substituted metal ions were removed from the active site during titration with the chelator 2,2-dipyridyl.