Tigecycline, a tetracycline derivative, shows atypical plasma protein binding behavior. The unbound fraction decreases with increasing concentration at therapeutic concentrations. Moreover, uncertainty exists about the magnitude of tigecyline's protein binding in man. Unbound fractions between 2.5% and 35% have been reported in plasma from healthy volunteers, and between 25% and 100% in patients, respectively. In the present study, the protein binding of tigecycline has been investigated by ultrafiltration using different experimental conditions. Whereas temperature had only a marginal influence, the unbound fraction at 0.3/3.0 mg/L was low at pH 8.2 (9.4%/1.9%) or in unbuffered pooled plasma (6.3%/1.2%), compared with plasma buffered with HEPES to pH 7.4 (65.9%/39.7%). In experiments with phosphate buffer and EDTA, the concentration dependency was markedly attenuated or abolished, which is compatible with a cooperative binding mechanism involving divalent cations such as calcium. The unbound fraction in clinical plasma samples from patients treated with tigecycline was determined to 66.3 ± 13.7% at concentrations0.3 mg/L compared with 41.3 ± 16.0% at1 to5 mg/L. To summarize, tigecycline appears to be only moderately bound to plasma proteins as determined by ultrafiltration, when a physiological pH is maintained.