The cause of the activity loss of alternative pathway of electron transport in mitochondria of the yeast Candida lipolytica has been investigated. Incubation of cyanide-resistant mitochondria at 25 degrees was shown to cause the loss by mitochondria of their ability to oxidize substrates in the presence of 1 mM cyanide. This suggests that in the course of incubation the alternative pathway loses its activity. Repeated washing of mitochondria with a solution containing 2,5 mM EDTA inhibits, while Ca2+, Mn2+, Cu2+ and Zn2+ (but not Sr2+) enhance the process of the activity loss of the alternative pathway. The loss of the cyanide-resistant respiration is also observed during incubation of mitochondria in the presence of phospholipases A, C and D or lysolecithin. In all cases studied the reactivation of the cyanide-resistant respiration of mitochondria is attained by addition of azolectin. The loss of cyanide-resistant respiration is accompanied by the activity reduction of the main respiratory chain, which is restored by addition of cytochrome c and Mg2+. These data indicate that the activity loss of the alternative pathway is not related to inactivation of any components in the alternative pathway itself or in the main respiratory chain. The most probable cause of the activity loss in the destruction of reducing equivalents in the alternative pathway of a donor as a result of a break of the structural entity of the internal membrane of mitochondria due to the detersive action of the phospholipid lysoforms produced either by endogenic or exogenic phospholipases.