Background and Aims: Interleukin-17 (IL-17) is a T-cell-derived cytokine that may play an important role in the initiation or maintenance of the pro-inflammatory response and has recently been found to stimulate osteoclastic resorption. The purpose of the present study was to determine the presence of IL-17 in gingival crevicular fluid (GCF) samples and in the culture supernatants of gingival cells from patients with chronic periodontitis. Method: GCF samples were collected during 30s from two sites in 16 patients from periodontally affected sites (probing depth ≥ 5 mm, attachment loss ≥ 3 mm). The comparison with healthy controls was carried out by collecting GCF samples from eight healthy volunteers. GCF was collected using a paper strip and ELISA was performed to determine the total amount of IL-17. Supernatant cellular cultures of gingival cells were obtained from periodontal biopsies taken from 12 periodontitis patients and from eight healthy control subjects during the surgical removal of wisdom teeth. Spontaneous and phytohaemagglutinin (PHA)-stimulated levels of IL-17 were determined by ELISA. Results: The total amount of cytokine IL-17 was significantly higher in the periodontitis group than the control group (45.9 versus 35.6 pg, p = 0.005). Significantly higher GCF volume and amount of total proteins were obtained from periodontitis patients as compared with control subjects (0.98 versus 0.36 μl, p = 0.0005; 0.12 versus 0.05 μg, p = 0.0005, respectively). A higher concentration of IL-17 was detected in culture supernatants from periodontitis patients compared with healthy subjects, either without stimulation (36.28 ± 8.39 versus 28.81 ± 1.50 μg/ml, p = 0.011) or with PHA stimulation (52.12 ± 14.56 versus 39.00 ± 4.90 μg/ml, p = 0.012). Treatment with PHA induced a significant increase in the production of IL-17 in healthy subjects and periodontitis patients (p = 0.001 and 0.003). Conclusions: The total amount of cytokine IL-17 in GCF samples and in the culture supernatants of gingival cells are significantly increased in periodontal disease.