Galactinol synthase catalyzes the first committed step in the biosynthesis of raffinose sugars. Previous attempts to purify the enzyme have proven difficult and have resulted in low quantities of unpurified enzyme. Galactinol synthase was purified 752-fold from mature zucchini (Cucurbita pepo L. cv Burpee Hybrid) leaves using sequential liquid chromatography on DE 52, Octyl-Sepharose CL-4B, and Sephacryl S-200. This isolation scheme resulted in an 18.6% recovery of the initial activity. The purified enzyme had a specific activity of 23.3 micromoles per minute per milligram protein, a pH optimum of 7.5, and the activity was enhanced by dithiothreitol and MnCl2. The enzyme was only half as active with MgCl2 as with MnCl2. Na+, K+, and Ca2+ cations had little effect on the enzyme activity, while Co2+, Zn2+, Cu2+, and Fe3+ cations were strongly inhibitory at 10 millimolar concentrations. Purified galactinol synthase bound specifically to the substrates myo-inositol and UDP-galactose (Km = 6.5 and 1.8 millimolar, respectively), while exhibiting little affinity for an alternative glycosyl donor (UDP-glucose) or inositol epimers (epi- and scyllo-). Ten millimolar concentrations of UMP, UDP, UTP, AMP, ADP, ATP, NAD+, NADH, NADP+, UDP-xylose, and UDP-mannose, or 20 millimolar sucrose, talose, galactose, glucose, xylose, and melibiose exhibited various degrees of inhibitory effects. Twenty millimolar stachyose, raffinose, fructose, and mannose, and 10 millimolar UDP-glucuronic acid and UDP-galacturonic acid had little or no effect on the enzyme activity. The purified galactinal synthase is a monomer of Mr 42,000 with an isoelectric point of 4.1.