Diverse interstitial lung diseases (ILD) demonstrate mesenchymal infiltration by an abundance of activated mast cells whose role in parenchymal fibrogenesis remains unclear. Since mast cells differentiate in a dynamic, tissue-specific manner via signals transduced by c-Kit receptor, we examined the effect of ILD microenvironments on c-Kit expression and metalloproteinase phenotypes of mesenchymal mast cell populations. Immunohistochemical and flow cytometric analyses characterized surface expression of c-Kit on mast cells in tissues obtained from patients with idiopathic pulmonary fibrosis, systemic sclerosis, sarcoidosis, and lymphangioleiomyomatosis, thus identifying a unique immunophenotype not shared by normal lung mast cells. Isolation of c-Kit+/FcεRI+/CD34- mast cells via immunocytometric sorting of heterogeneous cell populations from mechanically disaggregated lung tissues permitted analysis of gene expression patterns by two-step real-time polymerase chain reaction. Transcriptional profiling identified expression of c-Kit and the neutral serine proteases, tryptase and chymase, establishing the identity of sorted populations as mature mast cells. Mast cells harvested from ILD tissues demonstrated characteristic metalloproteinase phenotypes which included expression of matrix metalloproteinase (MMP)-1 and a disintegrin and metalloproteinase (ADAM)-9, -10, and -17. Immunohistochemical co-localization guided by gene profiling data confirmed expression of chymase, MMP-1, and ADAM-17 protein in subpopulations of mast cells in remodelling interstitium. Gene profiling of harvested mast cells also showed increased transcript copy numbers for TNFα and CC chemokine receptor 2, which play critical roles in lung injury. We conclude that ILD microenvironments induce unique c-Kit receptor and metalloproteinase mast cell phenotypes. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.