Salicylic acid (SA) is known to induce alternative pathway respiration by activating expression of the alternative oxidase gene. In the present study we report a rapid mode of action by SA on plant mitochondrial functions. SA at concentrations as low as 20 μM induced inhibition of both ATP synthesis and respiratory O2 uptake within minutes of incubation in tobacco (Nicotiana tabacum) cell cultures. Biologically active SA analogs capable of inducing pathogenesis-related genes and enhanced resistance also caused rapid inhibition of ATP synthesis and respiratory O2 uptake, whereas biologically inactive analogs did not. Inhibition of ATP synthesis and respiratory O2 uptake by SA was insensitive to the protein synthesis inhibitor cycloheximide, but was substantially reduced by the antioxidant N-acetylcysteine, suggesting a possible role for reactive oxygen species in the inhibition of mitochondrial functions. With exogenous NADH as the respiratory substrate, mitochondria isolated from SA-treated tobacco cell cultures were found to have normal capacities for both ATP synthesis and respiratory O2 uptake; direct incubation of isolated mitochondria with SA had no significant effect on these mitochondrial functions. These results indicate that (a) the respiration capacities of isolated mitochondria do not correspond to the in vivo respiration activities in SA-treated cell cultures and (b) the SA-induced inhibition of respiration in tobacco cell cultures may involve other components that are not present in isolated mitochondria. Given the recently demonstrated roles of mitochondria in plant disease resistance and animal apoptosis, this rapid inhibition by SA of mitochondrial functions may play a role in SA-mediated biological processes, including plant defense responses.