We have previously shown that leukotriene D 4 (LTD 4), a known pro-inflammatory mediator, induces increased survival and proliferation of intestinal epithelial cells. In this study we examined whether LTD 4 functions via activation of the transcription factors NFκB and AP-1, which are potent inducers of mitogenesis. We found that the NFκB inhibitory protein IκBα was not degraded upon LTD 4 stimulation. Furthermore, nuclear translocation of the classical p65 or alternative p52 subunits of NFκB was not observed. Accordingly, LTD 4 stimulation failed to induce NFκB transcriptional activity. Instead we found that LTD 4 induced phosphorylation of c-Jun-N-terminal kinase (JNK) and transcriptional activity of AP-1, which could be reduced by a JNK inhibitor. Moreover, LTD 4 induced cell proliferation, and this effect was also blocked upon addition of a JNK inhibitor. Our findings show for the first time that JNK/AP-1 but not NFκB is a downstream target of LTD 4 in intestinal epithelial cells, suggesting that AP-1 is an important mediator of LTD 4-induced mitogenic effects.