In the small intestinal segment perfusion model the effect of osmolality on net fluid absorption in enterotoxigenic Escherichia coli (ETEC)-infected and non-infected small intestinal segments of piglets was investigated. In ETEC-infected segments net fluid absorption was reduced. Lowering the osmolality from 375 to 150 mOsmol/l by reducing sodium chloride concentrations increased net fluid absorption. There was a linear relationship between osmolality and net fluid absorption for both non-infected and ETEC-infected segments. Below 150 mOsmol/l the inverse relationship between osmolality and net fluid absorption disappeared. Substitution of sodium chloride with mannitol decreased net fluid absorption since mannitol cannot be actively absorbed. In ETEC-infected segments perfused with oral rehydration solution net fluid absorption significantly increased compared to a sodium chloride solution of similar osmolality, probably because of the high glucose concentration. Supplying hypotonic sodium chloride fluids to piglets can be beneficial in replenishing water and electrolyte losses in case of infectious diarrhoea.