&NA;The effects of temperature on binding characteristics of phenytoin (PHT) to serum proteins were determined in adult patients with epilepsy. Serum samples examined in the study were obtained from 47 adult patients (29 men, 18 women) with epilepsy on PHT monotherapy. Ages ranged from 18 to 64 years (mean [SD], 36.8 [12.1] years). Protein binding of PHT was evaluated by ultrafiltration under current laboratory routine conditions (25 ± 3°C) or at a temperature of 37°C. The in vivo binding parameters of PHT to serum proteins were determined using a binding equation derived from the Scatchard equation for a one-site binding model. Significant differences were observed in serum concentrations of unbound PHT between paired data (P< .05). The mean association constants (K) of PHT to serum proteins are 0.009 L &mgr;mol−1at 25 ± 3°C and 0.003 L &mgr;mol−1at 37°C, whereas mean total concentrations of binding sites [n(Pt)] are 1215 &mgr;mol L−1for 25 ± 3°C and 2263 &mgr;mol L−1for 37°C. Significant differences were observed in binding characteristics of PHT to serum proteins between the data determined in different conditions of ultrafiltration (P< .05). Our study confirms that binding affinity for PHT-serum protein interaction is approximately 67% lower at 37°C than at 25 ± 3°C, and, consequently, binding potential [K •n(Pt)] is approximately 38% lower at 37°C than at 25 ± 3°C.