OBJECTIVE To investigate glucocorticoid uptake in auditory hair cells following transtympanic versus systemic administration of dexamethasone. METHODS Controlled experimental study. METHODS Translational science experimental laboratory. METHODS Swiss-Webster mice were injected with dexamethasone via transtympanic or systemic administration. At 1, 6, or 12 hours post-injection the temporal bones were harvested. After cryosectioning, immunohistochemical staining was performed using an antibody for dexamethasone. RESULTS Dexamethasone labelling was greatest at 1 hour. Inner hair cells demonstrated much higher steroid uptake than outer hair cells. Both transtympanic injection and high-dose systemic administration resulted in strong dexamethasone labelling of hair cells, and a decreasing basal-to-apical gradient of hair cell fluorescence intensity was observed. Systemically delivered dexamethasone was rapidly eliminated from the inner ear, demonstrating mild labelling after 6 hours and none after 12 hours. In contrast, the mice receiving transtympanic injection had persistent moderate intensity fluorescence at 6 and 12 hours post-injection. CONCLUSIONS There is similar uptake of dexamethasone by auditory hair cells after transtympanic and high-dose systemic delivery. Novel findings include the presence of a decreasing basal-apical gradient of steroid uptake, and demonstration of greater affinity of inner hair cells for dexamethasone compared to outer hair cells. In this animal model transtympanic injection resulted in prolonged steroid uptake. These findings help further our understanding of the pharmacokinetics of steroids in the cochlea, with a focus on auditory hair cells.