Objective: To investigate whether nerve growth factor (NGF) alters glutamate expression in sensory fibres and glutamate concentration in the masseter muscle of female rats. Methods: Ten female rats were injected with NGF (25 µg/ml, 10 μl) and vehicle into the right and left masseter muscles, respectively. Immunohistochemistry and microdialysis were performed after 3 days to evaluate glutamate expression and concentration in the muscle. Results: The frequency of expression of glutamate in the nerve fibres innervating the masseter muscle was significantly greater 3 days after NGF (56 ± 5%) than after vehicle (39 ± 5%) injection. The majority of fibres co-expressed the neuropeptide substance P (SP); a marker for sensory afferent fibres. There was no effect of NGF on the expression of the excitatory amino acid transporter type 2 (EAAT2). In the microdialysis experiment, mean interstitial glutamate concentration on the vehicle side (21.6 ± 9.8 µM) was not significantly different from that on the NGF side (16.2 ± 9.2 µM). Conclusion: These results suggest that, in part, NGF increases the mechanical sensitivity of the masseter muscle by increasing glutamate expression in the sensory nerve endings in the muscle. This effect was local to the site of the NGF injection, as it was only detectable through immunohistochemistry, but not by microdialysis. Keywords: glutamate, nerve growth factor, sensory fibre, temporomandibular disorders