Epidermal Growth Factor (EGF) has been identified as playing a critical role in the wound healing process. The objective of this study is to investigate the role that EGF plays in rat tympanic membrane (TM) wound healing using two techniques, microarray and immunohistochemistry. The tympanic membranes of rats were perforated using a sterile needle and sacrificed at time points during 2 weeks following perforation. The normalized signal intensities at the time points for EGF and associated genes are presented. The rat EGF mRNA did not change significantly between time points. Five associated proteins, including heparin-binding EGF-like growth factor were found to be differentially expressed above a two fold threshold at 12 h following perforation. EGF staining was found at low levels in the uninjured TM. Levels of EGF staining increased at 24 h in the basal keratinocyte layer, became diffusely elevated in the specimen at 36 h, before a second peak in staining of the keratinocyte layer at Day 4. The staining of EGF corresponds to its multiple roles in TM wound healing.