Objective: In earlier studies, the resonant frequency (RF) of the middle ear has been investigated in a number of different animals. However, no study has so far specifically addressed the measurement of RF in rats. With this in mind, in our study, multifrequency tympanometry (MFT) was performed on rats and RF value measurements were taken. The aim was to ascertain the normal values for rats used in our university experiments and to provide guidance for further research to be undertaken in this area. Materials and Methods: For the study, 16 male and 16 female adult Sprague Downey rats (32 animals, 64 ears) aged 10 months old and in healthy condition were used. MFT at 226Hz frequency and above was performed on all the rats after they had been anaesthetised. In the first instance tympanograms were recorded using the 226Hz probe tone, following which MFT measurements were performed. RF values for the middle ear were determined in the range 200-2000Hz. Results: For male rats, the mean RF value was 426.56 (± 193.01 Hz), whilst for female rats the mean was 496.88 (± 132.55) Hz. When the values obtained for the male and female rats were compared, no statistically significant difference was observed between the two groups (p=0.061). Accordingly, the mean RF value for the rats as a whole (obtained for 64 ears) was calculated to be 461.7 (± 168.02) Hz. Conclusion: In this research, RF values for the middle ear of rats has been demonstrated to be measurable. However, further research is needed into the relationship between RF and the characteristics of the middle ear in rats.