This study uses a cross-sectional design to examine the endothelial and inducible nitric oxide synthase (eNOS and iNOS, respectively) levels of gingival tissue. Fifteen subjects, 10 female and 5 male individuals (aged 14.6-21.2 years; mean 17.4 +/- 1.8 years), who needed extraction of the four first premolars for orthodontic reasons and who had indications for a gingivectomy were enrolled in the study. In each patient, two maxillary/mandibular premolars were extracted, and two months later an orthodontic appliance was placed in the same arch. A canine undergoing treatment for distal movement served as the test tooth (TT), whereas its contralateral canine was used as the control tooth (CT). The CT was included in the orthodontic appliance but was not subjected to the orthodontic force. Two weeks after the orthodontic appliance placement, clinical data consisting of the presence of supragingival plaque, bleeding on probing, and probing depth were collected from each experimental tooth. Immediately after, gingival tissue was collected from the distal aspect of each TT and CT for immunohistochemistry, messenger RNA reverse transcription by polymerase chain reaction, and Western blot analysis for both eNOS and iNOS. The results showed that no differences in clinical conditions occurred between the experimental teeth. On the contrary, both the eNOS and iNOS levels and the expression of the TTs were significantly greater than those of the CTs (all comparisons significant to P < .01). Our results indicate a role for gingival eNOS and iNOS during the early phases of orthodontic treatment in humans.