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Vesicular secretion of macromolecules has recently been described in the basidiomycete Cryptococcus neoformans, raising the question as to whether ascomycetes similarly utilize vesicles for transport. In the present study, we examine whether the clinically important ascomycete Histoplasma capsulatum produce vesicles and utilized these structures to secrete macromolecules. Transmission electron microscopy (TEM) shows transcellular secretion of vesicles by yeast cells. Proteomic and lipidomic analyses of vesicles isolated from culture supernatants reveal a rich collection of macromolecules involved in diverse processes, including metabolism, cell recycling, signalling and virulence. The results demonstrate that H. capsulatum can utilize a trans‐cell wall vesicular transport secretory mechanism to promote virulence. Additionally, TEM of supernatants collected from Candida albicans, Candida parapsilosis …