The objectives of the present study were to determine the effect of nicotine on MAPK signaling and on the proliferation of AR42J cells, as well as to assess the relationship between MAPK activation and exocrine secretion in these cells. AR42J cells were incubated with nicotine and analyzed for the activation of MAPK by Western blotting, using their respective antibodies and confirmed by immunohistochemistry. The effect of nicotine on cell proliferation was determined by spectrophotometric method, and cell function was assessed by CCK-stimulated amylase release into the culture medium. Nicotine at a dose of 100 µM induced phospho-ERK-1/2 activation maximally in 3 minutes as compared to untreated cells. Furthermore, immunofluorescence study confirmed nicotine-induced increase in translocation of phospho-ERK-1/2 to the nucleus. Activation of phospho-ERK-1/2 was inhibited by an ERK-1/2 pathway inhibitor but not by a nicotine receptor antagonist. At the same dose, there was significantly enhanced proliferation of AR42J cells until 72 hours without toxic effect, as percent of lactate dehydrogenase (LDH) release remained unchanged. Other MAPK, JNK-1/2 and p38-MAPK were not affected by nicotine treatment. At a nicotine dose of 100 µM, the CCK-stimulated release of amylase was maximal at 6 minutes, and while a nicotinic receptor antagonist inhibited this response, it was not inhibited by the ERK-1/2 pathway inhibitor. We conclude that nicotine treatment induced activation of ERK-1/2 and increased the proliferation of AR42J cells. The data further indicates that MAPK signaling by nicotine is independent of the secretory response.