Ragweed (Ambrosia artemisiifolia), the major cause of late summer hay fever (allergic rhinitis) in the United States and Canada, is clinically the most important source of the seasonal aeroallergens. A novel endopeptidase was extracted from the pollen of this plant and purified by a series of column chromatographic steps. It has a molecular mass of 82 kDa according to gel filtration and SDS-polyacrylamide gel electrophoresis and a pH optimum near 9.0, and its activity is unaffected by chelating or reducing agents. A 17-amino acid amino-terminal sequence of this protein showed no similarity with any other proteases. The enzyme was inhibited by diisopropyl fluorophosphate, a general serine class inhibitor, and more specifically N- p-tosyl-L-phenylalanine chloromethyl ketone, a chymotrypsin-like proteinase inhibitor. Various synthetic substrates were efficiently cleaved with a strong preference for Phe in the P1 and P3 position and Pro in the P2 position. This specificity was confirmed through inhibition studies with both peptidyl chloromethyl ketone and organophosphate inhibitors. In addition to synthetic substrates, the neuropeptides, vasoactive intestinal peptide and substance P, which are required for normalized lung functions, were also rapidly hydrolyzed. Activity toward protein substrates was not detected with the exception of the inactivation of α-l-proteinase inhibitor, which occurred through cleavage within the reactive site loop. These results indicate that the purified enzyme is a novel endopeptidase, which may be involved in both the degradation of neuropeptides and the inactivation of protective proteinase inhibitors during pollen-initiated allergic reactions.