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Muscle tenderness and pain during movements are prominent symptoms associated with persistent jaw muscle pain. However, there is virtually no information on how trigeminal neurons respond to jaw movements (JM) or muscle palpation in the presence of muscle tissue injury or myositis. In this study, we investigated the effects of innocuous JM in the presence of acute masseteric inflammation on postsynaptic responses in the trigeminal brainstem nuclei by examining the expression of c-fos. In one group of rats, unilateral injections of an inflammatory substance, mustard oil (MO: 20%, 25 μl) were made into a masseter muscle. In another group, controlled and systematic JM were provided following MO injection. Three additional groups of rats were used to control for anesthetic, JM, and injection procedure. MO injected in the masseter muscle induced a high level of Fos protein expression in four principal trigeminal regions: the subnucleus caudalis (Vc), the ventral and dorsal regions of the Vc/Vi (subnucleus interpolaris) transition zone, and the paratrigeminal nucleus (PTN). Movements following MO injection consistently produced a significantly greater level of Fos expression in all these areas, especially in the Vc/Vi transition region and caudal Vc on the ipsilateral side. Importantly, movements also induced a significantly greater level of Fos expression in the caudal Vc on the contralateral side. The present results provide the first documentation that innocuous JM in the presence of muscle inflammation significantly increase the MO-induced c-fos expression in the trigeminal brainstem nuclei, which may explain the greater pain experienced during movement of inflamed or injured muscles.