The gastrointestinal tract is exposed to environmental insult as a result of food intake or in pathological conditions such as diarrhoea, and is therefore protected by the mucus layer. As part of it, trefoil peptides (TFFs) are able to modify the visco-elastic properties of the mucus, protect against experimental ulceration, and promote repair of the epithelia. We investigated, using transient reporter gene assays and RT-PCR in the gastric carcinoma cell line MKN45 and colon carcinoma cell lines LS174T and HT29, whether ethanol and osmotic changes can modify transcriptional activity of TFFs. In a mild hypotonic environment (200 mosmol/l) all three TFF genes were up-regulated by at least a factor of 2. In hypertonic medium (400 mosmol/ll), TFF1 and TFF3 were down-regulated, whereas TFF2 was up-regulated by elevated concentrations of sodium or chloride in MKN45. Raising the osmolality by ethanol resulted in an up-regulation of TFF3 in both colon cell lines but not in the gastric cell line. We conclude that alteration in TFF gene expression is a response of gut epithelia to deal with osmotic forces and ethanol.