The oxidation of choline by both freshly prepared and aged rat liver mitochondria is inhibited by amytal. Whereas rotenone inhibits choline-cytochromec reductase only in the case of freshly prepared mitochondria, the extent of inhibition is influenced by preincubation, but the inhibition is not secondary to the inhibited oxidation of betaine aldehyde, the product of choline oxidation. Evidence shows that rotenone is able to inhibit the swelling of rat liver mitochondria and the inhibition of choline-cytochromec reductase by rotenone is related to the inhibition of mitochondrial swelling. Nine inhibitors of choline dehydrogenase have been reported. Among those, some belong to the category of acetylcholine esterase inhibitor. In view of the structure of those inhibitors, it seems quite likely that there is an anionic site at the active center of choline dehydrogenase. Purification of choline dehydrogenase in its native form has been accomplished by solubilization with Lubrol WX, hydroxyapatite, and DEAE-Sepharose chromatography and sucrose gradient ultracentrifugation. The preparation is pure as judged by SDS-PAGE and Ultrogel AcA 34 column chromatography. The molecular weight determined by SDS-PAGE is approximately 61,000. There is 0.23 mg phospholipid/mg protein and the Stokes' radius of protein-Lubrol-phospholipid mixed micelles is about 59 A.