Transseptal fibers of the periodontal ligament are thought to play a role in relapse after orthodontic derotation. The aim of this study was to compare the location of the attachment site and the arrangement of the transseptal fibers in rotated emerged and non-rotated emerged teeth. In five dogs, unilaterally, the first upper premolars were replaced into a rotated position, before emergence. Histological evaluation showed that transseptal fibers developed in the experimental as well as in the control specimens. In all cases, transseptal fibers bridged the shortest distance between two adjacent teeth. It was concluded that the attachment site of the transseptal fibers was not determined by the tooth anatomy itself, but by the tooth position and its orientation in the dental arch during transseptal fiber development.