To explore mechanisms whereby Malpighian keratinocytes can transdifferentiate into an intestinal-like epithelium, as observed in the early steps of Barrett's esophagus (BE) development, long-standing cultures of esophageal keratinocytes derived from normal mouse esophageal explants were developed. These cells were able to form multilayers and to differentiate on filter support by the formation of differentiated layers of basal cells (cytokeratine 14 positive) on which secondary suprabasal cell layers (cytokeratine 4 positive) spontaneously developed. Thus, these cultured cells, referred to as P3E6, reproduced, at least in part, the proliferation and stratification pattern existing in the normal esophagus. Because chronic exposure to acid pH is known to be a critical factor for BE development, culture medium at pH 3.5 was added into the apical chamber of cell cultures. This led to a decrease in the overall number of cells but it did not affect cell proliferation. Furthermore, external acid environment triggered expression of the GFP reporter gene fused downstream of the cdx2 intestinal homeogene regulatory sequences in P3E6 transfected cells. Expression of the endogenous CDX2 protein, detected by western blot and immunocytochemical analysis, correlated with promoter activation. These findings demonstrate that chronic exposure of esophageal keratinocytes to acid pH induces transcription of cdx2, an intestinal specific homeobox gene known to play a critical role in the differentiation and maintenance of intestinal epithelial functions. The results suggest that chronic acid exposure can modify the fate of P3E6 esophageal keratinocytes towards an intestinal program. This can be a key step in the development of intestinal metaplasia often observed in esophagus-cardia junction.