Ethanol is a well established irritant inducing inflammation in gastric mucosa, but the effects at the cellular level remain unclear. This study investigates NF-B activation in gastric mucosal cells by ethanol and assesses the effects of heat shock pretreatment in this ulcerogenic situation. Rat gastric mucosal epithelia (RGM-1) were exposed for different time periods to ethanol. Heat shock was induced by incubating the cells at 42°C for 1 h prior to the experiments. For evaluation of NF-B activation the nuclear fraction of the cell lysates was analyzed with electrophoretic mobility shift assay or with ELISA based assay. Caspase-3 (promoter of apoptosis) activity was measured with time resolved fluorescence based assay, cell viability with tetrazolium assay and cell membrane integrity with lactate dehydrogenase assay. Ethanol (1-5%) induced NF-B activation, reaching a maximum after 3 h and led also to moderately increased COX-2 expression. Heat shock pretreatment and intracellular calcium chelator, BAPTA , were able to inhibit ethanol induced NF-B activation. Heat shock pretreatment decreased ethanol induced caspase-3 activation, decreased cell membrane damage and retained cellular viability. Inhibition of NF-B activation by NEMO binding peptide or decreasing RelA expression or inhibiting COX-2 activity by CAY14040 promoted the effects of ethanol, such as increased caspase-3 activity and decreased cell viability. In conclusion, ethanol induces NF-B activation via a calcium dependent pathway and induces COX-2 expression. Inhibition of the NF-B activation or COX-2 activity potentiates apoptosis and cell damage induced by ethanol, suggesting a protective role for NF-B activation and COX-2 expression.