The mucosal upheaval (MU), where the mucosal wave starts and propagates upward, appears only when the vocal fold vibrates. The location of the MU histologically and the effect of changes in mean air flow rate (MFR) and vocal fold length on occurrence of the MU were studied in twelve excised canine larynges. The lower surface of the vocal fold was marked to serve as a landmark for subsequent study. Cricothyroid approximation was performed to lengthen the vocal fold. After taking high-speed pictures or recording stroboscopic images from the tracheal side, a small cut wound was made at the mark. This wound served to compare the position of the MU with the histologically identified location of the mark. The larynx was then sectioned in the frontal plane. Before lengthening the vocal fold, the MU occurred on the area where the lamina propria became thinner and where the muscular layer neared the epithelial layer. After lengthening the vocal fold, the MU actually shifted medially compared with its original position. The subglottic area surrounded by the bilateral MUs became longer and thinner. Whether or not complete glottal closure during a vibratory cycle was achieved did not alter these findings. In contrast, with a fixed vocal fold length the MU appeared more laterally as MFR increased, but, based on the relation with the mark, its location on the vocal fold did not change from its original position before increase of MFR.