We have previously identified mutations in the ELA2 gene, encoding neutrophil elastase, a serine protease of neutrophil granules, as the hereditary cause of cyclic hematopoiesis (CH) and severe congenital neutropenia (SCN). CH is an autosomal dominant human disease in which neutrophils and monocytes sinusoidally oscillate in opposite phase with 21 day frequency. SCN is a genetically heterogeneous human disorder typified by a promyelocytic arrest in the bone marrow, with severe peripheral neutropenia. Both diseases lead to opportunistic infection and an increased risk for acute myelogenous leukemia. Genetic linkage analysis was used to map the locus for CH to chromosome 19pter and heterozygous mutations were identified in ELA2 by positional cloning. As a candidate gene, mutations in ELA2 were also identified in most cases of SCN. From a total of 24 unique mutations in ELA2 detected among 41 different patients (7 mutations causing CH, 15 associated with SCN, 2 found in both patient populations, and 5 mutations occurring in individuals developing leukemia), we have now investigated the biochemical properties and intracellular processing of the mutant elastase protein through in vitro expression in cultured hematopoietic cells. We find that the mutations appear to be normally processed and routed to granules, collectively diminish activity toward the native substrate, and demonstrate a proclivity for alternate peptide substrates. We conclude that change of specificity mutations in a granule protease may be the basis for the spectrum of hereditary neutropenic and leukemic disorders in the “ela-opathies”.