Spinach leaf discs accumulated betaine when exposed to a mannitol solution of -20 bars. The accumulation was 12 micromoles per gram original fresh weight in a 24-hour period.Betaine-aldehyde dehydrogenase (EC 1.2.1.8) was assayed in various subcellular fractions prepared from spinach leaves, and it was found only in the soluble fraction. This cytosolic enzyme was purified 175-fold, and its properties were studied. The enzyme was relatively specific for betaine aldehyde as the substrate with an apparent K(m) value of 2.08 x 10(-4) molar. It also exerted activity on other aldehyde analogs tested, but with lower V(max) and higher K(m) values. The enzyme was relatively specific for nicotinamide adenine dinucleotide as the coenzyme, having an apparent K(m) value of 9.46 x 10(-6) molar; lower activities were observed when nicotinamide adenine dinucleotide phosphate or 3-acetyl pyridine adenine dinucleotide were tested as electron acceptors. The activity was enhanced by dithiothreitol and inhibited by p-chloromercuribenzoate, and the inhibition by p-chloromercuribenzoate was partially reversed by the subsequent addition of dithiothreitol. The activity was inhibited by high concentrations of NaCl and, to a lesser extent, proline. The equilibrium of the enzymic reaction was strongly in favor of betaine formation.The in vitro activity of the enzyme under optimal assay conditions was high enough to account for the amount of betaine accumulated under water stress conditions. The enzyme activity was the same in unstressed leaves and in leaves that had been water stressed for 24 hours.