The alternative oxidase (AOX) of plant mitochondria is encoded by the nuclear gene Aoxl. Sense and antisense D N A constructs of Nicotiana tabacum A o x l were introduced into tobacco, and transgenic plants with both increased and decreased levels of mitochondrial AOX protein were identified. Suspension cells derived from wild-type and transgenic plants were grown in heterotrophic batch culture. Transgenic cells with increased A O X protein had an increased capacity for cyanide-resistant, salicylhydroxamic acid-sensitive respiration compared to wild-type cells, whereas transgenic cells with decreased AOX protein had a decreased capacity for such respiration. Thus, genetic alteration of the level of A O X protein was sufficient to alter the capacity for electron transport through the alternative pathway. Under our standard growth conditions, "antisense" cells with dramatically reduced levels of AOX protein had growth and respiration rates similar to the wild type. However, whereas wild-type cells were able to grow under conditions that severely suppressed cytochrome pathway activity, antisense cells could not survive this treatment. This suggests that a critical function of A O X may be to support respiration when the cytochrome pathway is impaired. The much higher level of AOX protein in "sense" cells compared to the wild type did not appreciably alter the steady-state partitioning of electrons between the cytochrome path and the alternative pathway in vivo, suggesting that this partitioning may be subject to additional regulatory factors.Higher plants have two paths of mET from UQ to O2 (Day et al., 1980;Moore and Siedow, 1991). Electron transfer through the CP is coupled to ATP synthesis. The terminal oxidase (Cyt oxidase) is inhibited by CN. Electron flow from UQ through the AP is not coupled to ATP production. The terminal oxidase is called AOX and is inhibited by SHAM. Despite a great research effort, the functional role of the AP in nonthermogenic respiratory metabolism has not been clearly defined.Studies have shown that both the capacity of the AP to support O2 consumption and the activity of the AP in con-