Objectives/Hypothesis: The objective was to reveal the location of the neuromuscular junctions in a three-dimensional reconstruction of the human thyroarytenoid muscle within the true vocal fold. Study Design: Immunohistochemical analysis of serially sectioned human true vocal folds was performed, followed by reconstruction in three dimensions using computer imaging software. Methods: Six fresh human larynges from autopsy were harvested, fixed in formalin, and embedded in paraffin. Eight vocal cords were studied from these six larynges. Five-micron serial sections were collected throughout the entire vocal cord in an axial plane at 500-μm intervals. Immunohistochemical analysis was performed with anti-synaptophysin antibody. A computer-controlled imaging and reconstruction system was used to create a three-dimensional reconstruction from the serial sections and to represent the location of the clustered band of neuromuscular junctions within each true vocal fold. The vocal cord was divided into equal thirds from anterior to posterior for statistical analysis. Results: The most neuromuscular junctions (74%) we're located in the middle third, and the least (7%) were found in the anterior third. The difference in anterior-to-posterior distribution was statistically significant in all eight specimens by χ2 analysis (P < .001). Conclusion: The distribution of neuromuscular junctions is not random within the human thyroarytenoid muscle. Because neuromuscular junctions are most highly concentrated in a band within the mid belly of the muscle, botulinum toxin type A (Botox) injection in patients with spasmodic dysphonia should be targeted to this region.