Fluorescence and NMR relaxation studies have been performed on horse liver alcohol dehydrogenase (alcohol: NAD + oxidoreductase, EC 1.1.1.1) as a function of temperature. Observations of both the intrinsic protein fluorescence and the fluorescence of a noncovalently bound apolar probe, 2-(p-toluidinyl)naphthalene-6-sulfonic acid (TNS), indicate that a significant thermal transition occurs in the protein in the range of temperature 0-40 degrees C, and that there are different temperature-dependent forms of the enzyme. The transition between these forms is affected by the binding of specific ligands to the enzyme's active site. Time-resolved fluorescence studies of the two tryptophan residues in the enzyme suggest that this thermal transition occurs around tryptophan-314, which is buried near the intersubunit region. Binding of nucleotide to the enzyme causes a decrease in spin-lattice relaxation time, T1, which may result from a decrease in the number of water molecules bound to the protein. The observed results may be due to the interactions between the structural domains into which the monomer of the protein is folded.