To demonstrate that an uncoupling of respiration and phosphorylation, measured in vitro, reflects an in vivo situation, we badly need in vivo measurements of some uncoupling-linked parameters. The importance of this assertion is illustrated by studies of Barja and co-workers. A lower rate of H(2)O(2) production by mitochondria isolated from long-lived birds compared with short-lived mammals of the same body weight (see publications by Barja's and Sohal's groups) could be explained by (i) an in vivo difference or (ii) an in vitro artefact. In both cases, the reason for lower H(2)O(2) production may well be the same, i.e. a mild uncoupling of respiration in avian mitochondria showing lowered respiratory control. Again, this should be due to an in vivo operation of some bird-specific natural uncouplers (the first case) or stronger in vitro damage to the avian mitochondria during their isolation and incubation (the second). The latter possibility seemed more probable when Barja and co-workers revealed that the level of antioxidants in birds is lower than in mammals. However, further studies by the same group showed that the degree of unsaturation of fatty acids in birds is lower than in mammals, indicating a greater resistance of avian mitochondria to oxidative damage in vitro. Indeed, it was found that lipid peroxidation in isolated avian mitochondria occurs at a much lower rate than in mammals. More importantly, the in vivo level of peroxidation of lipids and proteins appears to be lower in birds than in mammals. Thus, it seems probable that longer lifespan of birds really does correlate with a slower rate of production of H2O2 by mitochondria in vivo.