S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) from rat liver was purified to homogeneity. The enzyme had a molecular weight of 188,000 and was composed of 4 subunits with a molecular weight of 47,000. The isoelectric pH of the enzyme was at 5.7. The Km values for S-adenosyl-L-homocysteine, adenosine, and DL-homocysteine were 15.2 microM, 1.05 microM, and 155 microM, respectively, at pH 7.2 and 25 degrees C. The enzyme showed a fluorescence with an emission maximum at 350 nm when excited at 280 nm. The protein fluorescence was partially quenched on addition of adenosine. The fluorescence quenching as a function of adenosine concentration indicated that the enzyme contained four binding sites for adenosine per molecule. The same data provided a value of 0.63 microM for the dissociation constant of adenosine. The enzyme possessed 4 mol of tightly bound NAD+/mol. The addition of adenosine to the enzyme caused the appearance of a peak at 327 nm in a concentration-dependent manner. The enzyme-bound NAD+ readily formed an adduct with bisulfite with the concomitant loss of enzyme activity. These data suggest that the bound NAD+ is involved in the catalytic mechanism of the enzyme.