A novel enzyme that catalyses the oxygen-dependent oxidation of 3-nitropropionic acid (3NPA) to malonate semialdehyde, nitrate, nitrite and H 2 O 2 has been purified from leaf extracts of the horseshoe vetch, Hippocrepis comosa , and named 3NPA oxidase. The enzyme is a flavoprotein with a subunit molecular mass of 36 kDa containing 1 molecule of FMN and exhibits little specificity for all nitroalkanes tested other than 3NPA (apparent K m 620 μ M). The maximum enzyme activity in vitro was expressed at pH 4.8 and was inhibited strongly by the products nitrate and nitrite. 3NPA oxidase activity was detected in green shoots, which also contain high concentrations of 3NPA, from plants grown with nitrate, ammonium or N 2 as sources of nitrogen. Enzyme activity was absent from roots and cell cultures, neither of which accumulate high levels of 3NPA.