Background: Gingival enlargement is one of the side effects associated with the administration of phenytoin. The mechanism by which phenytoin induces gingival enlargement is not well understood. This study was conducted to investigate the relationship between plasma and gingival crevicular fluid (GCF) phenytoin concentrations and the degree of gingival overgrowth in patients with similar gingival and plaque indices and also to determine the risk factors for gingival enlargement. Methods: Eighteen patients taking phenytoin in regular doses ≥6 months prior to the investigation participated in the study. Gingival enlargement was evaluated with two indices to score vertical and horizontal overgrowth. The gingival index (GI), plaque index (PI), gingival bleeding time index (GBTI), probing depth (PD), and clinical attachment level (CAL) were also evaluated. GCF and plasma phenytoin concentrations were determined by using high-performance liquid chromatography (HPLC). Results: There was no significant difference between responders and non-responders for PD, CAL, PI, GI, and GBTI. Phenytoin was detected in all of the GCF and plasma samples using the HPLC analysis method. The mean concentration of phenytoin in GCF was significantly greater than the concentration of phenytoin in plasma. No significant difference was observed for the concentration of GCF phenytoin between responders and non-responders. However, the concentration of plasma phenytoin was significantly higher in responders than non-responders. Conclusion: This study showed that plasma phenytoin level appeared to be risk factor for phenytoin-induced gingival overgrowth.