The regional distribution of S-adenosylhomocysteine hydrolase was determined in the rat brain. Small variations in enzyme activity between different regions were observed. Highest activity was found in hypothalamus and bulbus olfactorius, the least in pons and medulla. About 70% of the enzyme activity was recovered in the soluble fraction of the tissue homogenate and 25% was localized to the crude mitochondrial fraction. The corresponding values for lactate dehydrogenase were 40% and 50%, respectively. The small amount of enzyme (5%) sedimenting with the nuclear fraction could be explained by contamination of this fraction with soluble proteins and synaptosomes. Further separation of the crude mitochondrial fraction by discontinuous sucrose gradient centrifugation showed that most of the enzyme activity was localized to the synaptosomes, but a substantial amount was found in the top layer of the gradient. The relative specific activity of lactate dehydrogenase in the top layer was less than that of S-adenosylhomocysteine hydrolase. No time-dependent leakage of S-adenosylhomocysteine hydrolase from the synaptosomes could be demonstrated. After hypoosmotic treatment of the crude mitochondrial fraction and separation of this fraction on a discontinuous sucrose gradient, S-adenosylhomocysteine hydrolase and DOPA decarboxylase showed the same distribution in the gradient and were recovered in the cytoplasmic fraction.