Severe congenital neutropenia is a heritable human disorder characterized by neutropenia and acute myelogenous leukemia. We recently determined that the majority of cases result from de novo or autosomal dominantly inherited heterozygous mutations in ELA2, encoding neutrophil elastase. Neutrophil elastase is a chymotryptic serine protease localized in granules of neutrophils and monocytes and is the major target of inhibition of the serpin alpha(1)-antitrypsin. The mutations causing severe congenital neutropenia consist of amino acid missense substitutions, in-frame deletion, splice donor mutation producing a deletion, splice acceptor mutation causing insertion of novel residues, and protein truncating mutations of the carboxyl terminus resulting from nonsense substitutions and deletions leading to frameshifts. We have expressed 14 mutant forms of neutrophil elastase in vitro and have characterized their biochemical properties. The mutations have variable effects on proteolytic activity, eliminating the possibility that the disease results from haploinsufficiency. There is no evidence that the mutant enzymes are cytotoxic. The mutant enzymes retain vulnerability to inhibition by alpha(1)-antitrypsin, but demonstrate variable avidity for interaction with this serpin. Somewhat surprisingly, the mutant enzymes inhibit the wild type enzyme when both are coexpressed within the same cell, suggesting the potential to interfere with normal subcellular trafficking or post-translational processing.