Investigations were undertaken to evaluate which uraemic solutes decrease drug protein binding. This was done by performing HPLC-fractionation of uraemic biological fluids and studying the effect of addition of a lyophilisate of each fraction to normal plasma containing standard quantities of radiolabelled drugs. From a first study, based only on fractionation of uraemic ultrafiltrate with an HPLC-gradient mainly aimed at elution of hydrophilic compounds, hippuric acid appeared to be a major protein binding inhibitor for theophylline and phenytoin. The problem with this approach was that it did not include the compounds with the most substantial protein binding. Therefore, studies were planned to fractionate deproteinized uraemic sera, but first it was necessary to define which deproteinisation methods gave the highest yields of protein bound ligand. Heat denaturation was found to be one of the most effective deproteinisation methods. When a lyophilisate of uraemic serum, deproteinised by this method, was added to normal plasma, a higher capacity to displace theophylline from protein binding sites was found compared to the effect of an identical volume of an ultrafiltrate of the same samples. Fractionation of the deproteinised sample by HPLC revealed a larger number of fractions able to inhibit drug protein binding.