The unique ability of olfactory neurons to regenerate in vitro has allowed their use for the study of olfactory function, regeneration, and neurodegenerative disorders; thus, characterization of their properties is important. This present study attempts to establish the timeline of structural (protein expression) and functional (odorant sensitivity) maturation of human olfactory epithelial cells (hOE) in vitro using biopsy-derived cultured tissue. Cells were grown for 7 days; on each day, cells were tested for odorant sensitivity using calcium imaging techniques and then protein expression of each cell was tested using immunocytochemistry for proteins typically used for characterizing olfactory cells. Previous studies have shown that mature olfactory neurons in vitro attain a unique “phase-bright” morphology and express the olfactory marker protein (OMP). By day 3 in vitro, a variety of cells were odorant-sensitive, including both “phase-bright” and “phase-dark” cells that have previously been considered glial-like cells. The functional maturation of these hOEs appears to take place within 4 days. Interestingly, the emergence of an odorant sensitivity profile of both phase-bright and phase-dark cells preceded the expression of marker protein expression for OMP (which is expressed only by mature neurons in vivo). This structural maturation took 5 days, suggesting that the development of odorant sensitivity is not coincident with the expression of marker molecules that are hallmarks of structural maturation. These results have important implications for the use of hOEs as in vitro models of olfactory and neuronal function. © 2013 Wiley Periodicals, Inc.