The metabolism of [U-14C]-4-hydroxybutyric acid (GHB) was investigated in sonicates of mitochondria of rat heart, kidney and liver. The conversion of this precursor to 14C-organic acids was monitored and quantified by sequential liquid-partition chromatography on hydrated columns of silicic acid using a concave-upward gradient of 2-methylbutan-2-ol in chloroform. Sonicates of liver and kidney mitochondria, but not heart, readily converted [U-14C]-GHB to 14C organic acids via a pathway of conversion to 14C-succinic acid, followed by further metabolism through the tricarboxylic acid cycle. This conversion was facilitated by exogenous NAD+ and NADP+. No evidence for the beta-oxidation of GHB was obtained in any of the mitochondrial sonicates. Studies with exogenous non-labelled succinic semialdehyde indicated that this compound was an intermediate in the conversion of GHB to succinic acid.