Polyethylene glycol (PEG) has been used to increase the osmotic pressure of fluids used to cleanse the gastrointestinal tract. However, little is known about its osmotic activity. To investigate this activity systematically, solutions of PEG of differing molecular weights were made and subjected to measurement of osmolality by both freezing point depression and vapor pressure osmometry. Measured osmolality was increasingly greater than predicted from average molecular weight as PEG concentration increased. Measurement of sodium activity in NaCl/PEG solutions by means of an ion-selective electrode suggested that the higher than expected osmolality could be due in part to interactions that, in effect, sequestered water from the solution. Osmolality was consistently greater by freezing point osmometry than by vapor pressure osmometry. To determine which osmometry method reflected biologically relevant osmolality, normal subjects underwent steady-state total gut perfusion with an electrolyte solution containing 105 g/L of PEG 3350. This produced rectal effluent that was hypertonic by freezing point osmometry but isotonic by vapor pressure osmometry. Assuming that luminal fluid reaches osmotic equilibrium with plasma during total gut perfusion, this result suggests that the vapor pressure osmometer accurately reflects the biologically relevant osmolality of intestinal contents. We conclude that PEG exerts more of an osmotic effect than would be predicted from its molecular weight. This phenomenon may reflect interactions between PEG and water molecules that alter the physical chemistry of the solution and sequester water from the solution.