Polyethylene glycol has been in use for a number of years for the assessment of gut permeability. The methods so far employed are usually limited to polyethylene glycols in the low relative molecular mass range (up to M_r 1300). We developed a method for the simultaneous determination of gut permeability to M_r 400, M_r 1500, M_r 4000 and M_r 10000 polyethylene glycol, by applying a single oral dose of an appropriate mixture of these polyethylene glycols. After extraction from 24 h-urine, M_r 1500, M_r 4000 and M_r 10000 polyethylene glycol were quantified by size exclusion chromatography, while M_r 400 polyethylene glycol was determined by reversed phase chromatography. The detection limit of polyethylene glycol in the relative molecular mass range between M_r 1500 and M_r 10000 was found to be 0.2 mg/1 urine, and the detection limit of M_r 400 polyethylene glycol 5 mg/1 urine. Recovery of the polyethylene glycols (N = 6) were 86.6% (CV: 4.8%) for M_r 400, 94.1% (CV: 7.2%) for M_r 1500, 97.1% (CV: 5.5%) for M_r 4000 and 97.4% (CV: 5.6%) for M_r 10000. No significant difference was found between the excretion rates in 24 h-urine of M_r 400 and M_r 1500 polyethylene glycols in patients with Crohn's disease (M_r 400: 34.4 ± 5.5%; M_r 1500: 5.22 ± 2.27%; mean ± SEM, N = 10) and healthy controls (M_r 400: 33.6 ± 3.2%, M_r 1500: 1.09 ± 0.26%; N = 21). The excretion rate of M_r 4000 polyethylene glycol was markedly higher in patients with Crohn's disease (0.462 ± 0.177%) than in healthy controls (0.049 ± 0.012%, p < 0.05). M_r 10000 polyethylene glycol was detected in the urine of only one out of the 21 healthy controls, but in nine out of the ten patients with Crohn's disease (p < 0.001). These results suggest a change not in number but rather in selectivity in permeability of high M_r molecules.