The kinetic constants for the reversible adenosylhomocysteine hydrolase from rat liver have been determined. The Km values of the enzyme for S-adenosylhomocysteine, adenosine, and l-homocysteine were 12.3 micro M, 0.94 micro M, and 164 micro M, respectively. Under the specified conditions the Vmax for the synthetic reaction was 6.2 mumol/min per milligram, while the Vmax for the hydrolytic reaction was 0.72 mumol/min per milligram. l-Homocysteine acted as a mixed-type inhibitor of the hydrolytic reaction. Adenosine, a competitive inhibitor of both the synthetic and hydrolytic reactions (Ki = 1.2 +/- 0.2 micro M), was found to be a product of the reaction. In the absence of l-homocysteine about 0.8 nmol of adenosine was hydrolyzed to adenine and ribose per minute per milligram enzyme.