Perioral electrical stimuli cause inhibitory reflex responses in single motor-units (SMU) and surface electromyographic (EMG) recordings from voluntary contracted human jaw-closing muscles. Tonic experimental masseter pain has recently been shown to reduce the inhibitory reflex response in surface EMG recordings but the effect on SMU activity has not been described. In this study, motor-unit action potentials were recorded with wire electrodes inserted into the left masseter in eleven subjects. The subjects kept the SMU firing rate around 10 Hz by feedback. Ninety-nine electrical stimuli were applied sequentially to the left mental nerve with increasing stimulus delays in steps of 1 ms after the preceding motor unit action potential. The inhibitory reflex in SMU was recorded before, during and after infusion of hypertonic saline (5%) into the ipsilateral masseter muscle. Spike train data were used to calculate (1) the mean pre- and post-stimulus inter-spike-intervals (ISI) in all of the 99 trials, (2) cumulative changes in firing probability, and (3) estimation of the compound inhibitory post-synaptic potential (IPSP) in the masseter motoneuron. Tonic masseter pain did not change pre-stimulus SMU firing characteristics but the mean ISI for the first post-stimulus discharge (158.2+/-9.2 ms) was significantly decreased compared to the pre-pain (175.8+/-11.3 ms, P<0.05) and post-pain conditions (172. 6+/-11.6 ms, P<0.05). The post-stimulus firing probability was significantly increased and the relative amplitude of the estimated IPSP significantly decreased during tonic masseter pain compared to pre-pain and post-pain conditions. In conclusion, this study indicates that tonic masseter pain has a net excitatory effect on the inhibitory jaw-reflexes, which could be mediated by presynaptic mechanisms on the involved motoneurons.