A direct assay has been developed for succinic semialdehyde dehydrogenase in sonicates of human lymphocytes and Epstein-Barr Virus transformed cultured lymphoblasts. Enzyme activity was quantified by incubating cell extracts with uniformly labeled [¹⁴C]succinic semialdehyde and monitoring the conversion to [¹⁴C] succinic acid. Radiolabeled products were separated by liquid partition chromatography on hydrated silicic acid. Kinetic properties and requirements of succinic semialdehyde dehydrogenase in lymphoblast sonicates were investigated in order to determine optimal conditions for the direct assay. Enzyme activity was stimulated by dithiothreitol, ammonium and potassium ions and 0.1% Triton X-100. The concentrations for half maximal activation by ammonium and potassium were 5.2 and 13.7 mM respectively. The mean activity of succinic semialdehyde dehydrogenase in assays in which equimolar NADP ⁺ had been substituted for NAD ⁺ was 19 % of the activity of assays which contained NAD⁺. Substrate Michaelis constants were 21 and 30 μM for NAD⁺ and 26, 42 and 70 μM for succinic semialdehyde. The enzyme displayed a pH optimum between 8 and 9 and demonstrated a slight temperature activation between 37° and 45°C. A deficiency of succinic semialdehyde dehydrogenase activity was documented in cultured lymphoblasts derived from a patient with γ-hydroxybutyric aciduria.