Mitochondria treated with glycerol for several days at −15°C contract rapidly on the addition of adenosine triphosphate (ATP) and Mg++ When the contraction is induced by ATP and Mg++ optical density of a suspension of glycerinated mitochondria increases about 50 per cent or more and water extrusion or aggregation of internal structure also occurs in the mitochondria. Once controcted mitochondria reswell only gradually. The contraction was also initiated to a slight extent by inosine triphosphate in the place of ATP and by Mn++ or Co++ in the place of Mg++. The contraction is inhibited by azide, p-chloromercuribenzoate, arsenate and gramicidin. Sucrose depresses the reversal of swelling in intact mitochondria, but glycerinated mitochondria are differently affected by sucrose. ATPase activity in glycerinated mitochondria is essentially in parallel with the contractile activity, but both activities are different each other in the pattern of inhibitor action. Since the respiratory activity is restored on adddition of cytochrome c, there may be no cytochrome c in glycerinated mitochondria. Glycerinated mitochondria appear to suit for studying the mechanoenzyme system in mitochondria.