Objective: Determine the sequence of gross and histopathologic change to the normal middle ear (ME), tympanic membrane (TM), and external auditory canal (EAC) during spontaneous gerbilline cholesteatoma development. Study design and setting: Sixty-six gerbils were examined weekly and periodically sacrificed for analysis. Results: Cholesteatoma development followed this sequence: 1) slightly thickened pars flaccida (PF) without ME effusion, 2) thickened PF with ME effusion, 3) continuous buildup of EAC debris, and 4) complete occlusion of the lateral EAC. In the cholesteatoma group, keratinizing epithelium (KE) thickness was increased in all regions over normal controls and regional gradations in the TM remained the same. Conclusion: Spontaneous cholesteatoma formation was significantly associated with middle ear effusion, a failure to clear canal debris with resultant retrograde thickening of the EAC, PF, and then pars tensa. Significance: Inflammatory insult and alteration of the epithelial clearance mechanism appears to result in spontaneous cholesteatoma formation in gerbils as hypothesized for humans.