The sieving characteristics of the prototype drugs gentamicin (poorly protein bound) and phenytoin (highly protein bound) were measured in vitro using polysulfone capillary hemofilters similar to those used in clinical continuous arteriovenous hemofiltration. Plasma water, whole plasma, and whole blood were the solvent systems used with variable drug concentrations and solvent flow rates. Our results indicate that the sieving coefficients for both drugs can be accurately defined as the concentration in the ultrafiltrate divided by the concentration in the artery. This correlates with a more rigorously derived expression by Colton and Henderson (r = 0.98, P less than 0.00001 for both drugs) and avoids the necessity of measuring venous concentrations. Drug sieving in the three solvents followed expectations from known protein-binding data and was independent of solvent flow rate. For both drugs, at higher drug concentrations sieving increased in plasma, consistent with the saturation of protein-binding sites. In whole blood, sieving fell for both drugs with increasing drug concentrations, consistent with drug compartmentalization into red blood cells. Although these sieving changes with increasing drug concentrations were statistically significant, their clinical significance is doubtful. Phenytoin sieving in plasma was increased by the addition of free fatty acids, consistent with a protein-drug displacement effect. There appears to be a real, but small, effect of protein concentration polarization, protein-membrane and drug-membrane interactions on drug sieving. Our observations are consistent with the expectation that the major determinant of drug sieving is the extent of drug-protein binding.