During sustained isometric contraction of the jaw muscles, muscular fatigue causes a shift towards lower frequencies in the EMG frequency power spectrum. This shift is due to a reduction of myoelectric conduction velocity. The purpose of this study was to investigate the effect of surface electrode location on the estimates of frequency components, and to determine the appropriate frequency parameters of surface EMG which provided the best fit when comparing conduction velocity. Studies were performed in the masseter muscles of 8 healthy subjects. The myoelectric potentials were derived using 12 differential amplifiers from electrode arrays. The frequency parameters calculated were as follows: peak frequency, mean power frequency, cumulative power function (10, 25, 50 and 75%), median frequency ratio, low frequency ratio and measure H. As the results, following findings were obtained: 1. The high frequency content occurred at the region of end-plate zone and tendonous insertion of the muscle. 2. EMG spectra obtained during fatigue were shifted to lower frequencies for all regions except adjacent end-plate zone. 3. The relationships between conduction velocity and frequency parameters during fatiguing contractions were almost linear except peak frequency. 4. It was concluded that the location between distal end-plate and the tendon was "stable" zone for studying muscular fatigue.