PURPOSE. The density and three-dimensional localization of neuromuscular junctions (NMJs) of normal and botulinum toxin-treated normal adult rabbit and monkey extraocular muscles (EOMs) were analyzed. To demonstrate average myofiber length, randomly selected individual myofibers were reconstructed and compared with total muscle length. METHODS. Normal adult rabbit and monkey EOM and normal adult rabbit tibialis anterior were dissected in their entirety, frozen, sectioned longitudinally, and immunostained for NMJ localization. In addition, adult rabbit EOMs were injected with 5 U botulinum toxin, and NMJ density was determined after 2 weeks. NMJ locations for the three groups of EOM were reconstructed, and density of NMJ was determined. Individual myofibers were reconstructed from the orbital and global layers to determine mean fiber length. RESULTS. NMJS were dispersed throughout the entire length of all EOMs examined from adult rabbits and monkeys and were visualized by alpha-bungarotoxin staining and three-dimensional reconstruction of serial sections. In leg muscle, two relatively tight bands of NMJs were seen. Botulinum toxin significantly increased total NMJ density. Mean fiber lengths were 1.9 and 4.83 mm in the orbital and global layers, respectively, approximately 10% and 24% of the total origin-to-insertion muscle lengths. In addition, individual myofibers continuously changed their intrafascicular relationships over their lengths. CONCLUSIONS. The density and distribution of NMJs in normal EOMs are more extensive than previously described. Individual myofibers are significantly shorter than the tendon-to-tendon muscle length in both muscle layers. Botulinum toxin results in a doubling of NMJ density. NMJ localization in normal EOMs has ramifications for understanding eye movement control, but it is also important when surgical or pharmacologic intervention is used for the treatment of strabismus, nystagmus, or other eye muscle disorders.