Clinical studies have confirmed the adequate reproducibility of both centric occlusion and centric relation when used as reference positions during treatment; however, the reproducibility of the neuromuscular position has been found inadequate. This study evaluated the location and reproducibility of these three mandibular positions in relation to body posture, sitting and supine, and bilateral muscle activity before and after the insertion of a flat mandibular positioning device equilibrated to balance the muscle functions, as shown by two electromyography biofeedback instruments. Intraoral recordings were made in 11 young subjects with complete natural dentition. Acrylic resin clutches that supported a screw point in the maxillary arch and painted glass in the mandibular arch were used and positioned not to interfere with the occlusion. The distances of the screw scratch from two of the edges of the painted glass were used to measure the anteroposterior and mediolateral locations with a micrometer. The reproducibility was evaluated by measuring the scratch surface by measuring the weight of the print cutouts made from photographs of the scratches taken with a stereoscope. The location and reproducibility of centric occlusion and centric relation were not affected by body posture. A more precise posterior neuromuscular position was obtained in the supine position. The insertion of a mandibular positioning device did not affect centric occlusion but gave a more precise centric relation. Neuromuscular position became as precise as centric occlusion and was located anteroposteriorly between centric occlusion and centric relation.