Binding of phenobarbitone to human serum proteins was determined in vitro using ultrafiltration. At therapeutic concentrations of 10–600|μM, 63.51 ± 2.5% of the phenobarbitone was bound to total serum proteins. The binding kinetics were non-saturable and the plasma apparent affinity coefficient ( NK a ) was 0.791 ± 0.09 for normal serum and 0.664 ± 0.06 for chromatographed serum. Tobramycin and theophylline at therapeutic concentrations of 10 and 15 μg/ml, respectively, reduced NK a compared with the values found in normal serum. Calcium and especially magnesium at physiological concentrations of 2.5 and 5 mM, respectively, increased NK a compared with values obtained in chromatographed serum, whereas at supraphysiological concentrations of magnesium of 15 mM NK a was reduced. It is concluded that free phenobarbitone concentrations in serum can be modified by calcium, magnesium, tobramycin and theophylline, and this fact must be considered when studying enzyme induction and other properties of phenobarbitone.