1. The effects of 3-, 4- and 5-thia-substituted fatty acids on mitochondrial and peroxisomal beta-oxidation have been investigated. When the sulphur atom is in the 4-position, the resulting thia-substituted fatty acid becomes a powerful inhibitor of beta-oxidation. 2. This inhibition cannot be explained in terms of simple competitive inhibition, a phenomenon which characterizes the inhibitory effects of 3- and 5-thia-substituted fatty acids. The inhibitory sites for 4-thia-substituted fatty acids are most likely to be the acyl-CoA dehydrogenase in mitochondria and the acyl-CoA oxidase in peroxisomes. 3. The inhibitory effect of 4-thia-substituted fatty acids is expressed both in vitro and in vivo. The effect in vitro is instantaneous, with up to 95% inhibition of palmitoylcarnitine oxidation. The effect in vivo, in contrast, is dose-dependent and increases with duration of treatment. 4. Pretreatment of rats with a 3-thia-substituted fatty acid rendered mitochondrial beta-oxidation less sensitive to inhibition by 4-thia-substituted fatty acids.