The biliary clearances of fluid phase markers like erythritol and mannitol have been used to estimate canalicular bile flow. Larger fluid phase marker molecules like polyethylene glycol (PEG) 900 are excreted more extensively into bile, and it has been suggested that the biliary clearance of these give a more accurate measure of canalicular water flux than those of erythritol and mannitol. In this study, the biliary excretion of PEG 900 was compared with that of mannitol during choleresis induced by either sodium taurocholate or secretin. The biliary excretion of PEG 900 exceeded that of mannitol by a factor of 94. The biliary clearance of these markers was not influenced by secretin-induced choleresis. Using ion-exchange chromatography and fast atom bombardment mass spectrometry (FABMS) it was demonstrated that 26% of the PEG molecules are excreted into bile after oxidation to carboxylic acids, whereas sulphate conjugation is negligible. The majority of the PEG molecules (74%) were secreted unchanged, which supports the hypothesis of a mainly passive movement of PEG with the water flux into bile. FABMS showed an enrichment of larger PEG molecules in bile, which supports a previous finding that among differently sized PEGs the 1074-Da molecules have the highest biliary excretion.