An enzyme (M(r) 240,000) with high fatty acid alpha-oxidation activity has been purified from the fruit of cucumber (Cucumis sativus). The specific alpha-oxidation activity in the purified fraction was 370 nmol/min per mg protein determined as liberation of 14CO2 from [1-14C]palmitic acid. alpha-Oxidation activity was observed both in the 12,000 x g pellet and 150,000 x g pellet by differential fractionation of cucumber homogenate. The enzyme was purified about 220-fold to near homogeneity from a 12,000 x g fraction by solubilisation with Triton X-100R, ammonium sulphate precipitation, hydrophobic interaction and anion-exchange chromatographies and Superose 12 gel filtration. The molecular mass of the native enzyme was 240,000, and the major subunit molecular mass of 40,000 indicated an oligomeric structure.