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• Premise of the study: The Fungal Subcellular Ontology used in the Assembling the Fungal Tree of Life project is a taxon‐wide ontology (controlled vocabulary for attributes) designed to clarify and integrate the broad range of subcellular characters and character states used in higher‐level fungal systematics. As in the algae, cellular characters are important phylogenetic markers in kingdom Fungi. The Fungal Subcellular Ontology has been developed primarily to help researchers, especially systematists, in their search for information on subcellular characters across the Fungi, and it complements existing biological ontologies, including the Gene Ontology. • Methods: The character and character state data set used in the Assembling the Fungal Tree of Life Structural and Biochemical Database ( http://aftol.umn.edu ) is the source of terms for generating the ontology. After the terms were accessioned and defined, they were combined in OBO‐Edit file format, and the ontology was edited using OBO‐Edit, an open source Java tool supported by the Gene Ontology project. • Key results: The Fungal Subcellular Ontology covers both model and nonmodel fungi in great detail and is downloadable in OBO‐Edit format at website http://aftol.umn.edu/ontology/fungal_subcellular.obo . • Conclusions: The ontology provides a controlled vocabulary of fungal subcellular terms and functions as an operating framework for the Assembling the Fungal Tree of Life Structural and Biochemical Database. An ontology‐based design enhances reuse of data deposited in the Structural and Biochemical Database from other independent biological and genetic databases. Data integration approaches that advance access to data from the diversity of biological databases are imperative as interdisciplinary research gains importance. In this sense, the Fungal Subcellular Ontology becomes highly relevant to mycologists as well as nonmycologists because fungi interact actively as symbionts and parasites or passively with many other life forms.