We investigated the presence of hydroxyacid–oxoacid transhydrogenase (HOT), which catalyses the cofactor‐independent conversion of γ‐hydroxybutyrate (GHB) to succinic semialdehyde coupled to reduction of 2‐ketoglutarate (2‐KG) to D‐2‐hydroxyglutarate (D‐2‐HG), in human liver extracts employing [²H₆]GHB and 2‐KG as substrates. We measured incorporation of ²H in D‐[²H]2‐HG using GC‐MS analyses, providing evidence for HOT activity in humans. Kinetic characterization of HOT was undertaken in forward and reverse directions. We employed [²H₆]GHB and [²H₄]2‐KG as cosubstrates in order to develop a HOT activity assay in cultured human fibroblasts derived from patients with D‐2‐hydroxyglutaric aciduria. HOT activity was quantified in this system by the measurement of D‐[²H₅]2‐HG production. Fibroblasts derived from patients with D‐2‐hydroxyglutaric aciduria showed normal HOT activities. Our results provide the first demonstration and preliminary kinetic characterization of HOT activity in human tissues.