Animal models are frequently used to aid prediction of intestinal absorption in humans. However, there is little comparative quantitative information on species differences in paracellular permeation, which is an important route for oral absorption of small to medium-sized hydrophilic drug molecules. This study addresses this issue by comparing the molecular mass (MM) dependency in oral bioavailability between rat and dog of poly(ethylene glycol) (PEG), a polydispersed model mixture commonly used to characterize paracellular absorption, and of a series of eight D-peptides (based on D-phenylalanine). Fasted rats and dogs received PEG (400/900) and the D-peptides (MM 236-406 Da), orally and intravenously, with total 24-48 h urine collection to estimate oral bioavailability. After HPLC separation, the individual PEG oligomers and D-peptides were determined using radiometric detection, for radiolabeled material, and LC-MS, for unlabeled (PEG) material. All compounds were predominantly excreted unchanged following intravenous administration. After oral administration, the predominant peak in the radiochromatogram was unchanged material, indicating stability of the compounds in the gastrointestinal tract. A clear molecular mass dependency in oral bioavailability was seen with both series, but with absorption much greater in dog than rat. Thus, for PEG in rat, bioavailability decreased sharply from 79 to around 2% with increasing MM between 282 and 591 Da, and then tapered to around 1. 5% up to 1295 Da. Whereas in dog, bioavailability remained around 100% for oligomers up to 600 Da and then decreased quite sharply with increasing MM, tending to plateau around 13% beyond 900 Da. Likewise, for the d-peptides in rat, bioavailability decreased from 30 to 1% with increasing MM between 236 and 406 Da, whereas in dog it was 100%, declining to 16% over the same molecular range. This species difference appears to be due to a larger pore size and greater frequency of pores in the paracellular pathway of dog compared to rat. Furthermore, on the basis of comparison with literature data for PEG and selected drugs, rat would appear to be a better predictor than dog of absorption of hydrophilic compounds in human.