Abstract. Isolated mitochondria were maintained metabolically active and coupled, i.e., capable of respiratory control, for several hours at 250C. An increase in respiratory control generally occurred during the first 4-8 hr of incubation followed thereafter by a decline. Longevity is a dynamic function as evidenced by its dependency on substrate and cofactors, thiamine pyrophosphate, in particular. Magnesium was also essential to longevity; coenzyme A, bovine serum albumin, and reducing agents were not. The findings are discussed in terms of cytoplasmic-mitochondrial interrelationships and mitochondrial autonomy.Introduction. The quasi-autonomous nature of mitochondria is evidenced, in part, by their ability to conserve metabolically derived energy and their capacity to synthesize protein or other structural components in vitro.' However, the linearity of these functions has seldom been shown to exceed 1 or 2 hr2 and the resultant data, while highly valuable for point-in-time assessments of the organelles, can only be related to autonomy by extrapolation to in vivo conditions. More direct indications of autonomy would accrue if specific in vitro functions were shown to be essential for the maintenance of mitochondrial integrity and ultimately, for mitochondrial response to perturbations. Toward this goal we report that isolated mitochondria can remain active and phosphorylating for periods of up to 20 hr at 250C.Materials and Methods. Mitochondria were isolated from Bose and Bartlett pear fruit (Pyrus communis L.), and washed once, utilizing methods previously described.3 For survival at temperatures above 0C mitochondria (1-3 mg protein) and 3 ml of reaction mixture (as defined in Fig. 1) were placed in 20-ml beakers, covered with parafilm,