The biliary epithelium is exposed to mediators of inflammation such as bacterial endotoxin or lipopolysaccharide (LPS) in a variety of inflammatory conditions. These conditions are also characterized by cholangiocyte proliferation and a predisposition to malignancy. Furthermore, LPS can enhance the expression of interleukin-6 (IL-6), a known biliary mitogen. However, the effects of LPS on cholangiocyte proliferation or IL-6 secretion are unknown. Thus, our aims were to determine if LPS stimulates cholangiocyte proliferation by IL-6-dependent signaling pathways. H69 cells derived from normal human intrahepatic cholangiocytes proliferated in response to LPS. Cholangiocytes responded to LPS (and other inflammatory cytokines such as tumor necrosis factor alpha [TNF-alpha] and IL-1beta) by increased secretion of IL-6, which had a mitogenic effect on H69 cells. Preincubation with anti-IL-6 neutralizing antibodies inhibited LPS-induced proliferation. Furthermore, cholangiocytes possessed the IL-6 receptor complex subunits and intact signaling mechanisms leading to activation of signal transducers and activators of transcription (STAT) factors. Although both p38 and p44/p42 mitogen-activated protein kinases (MAPKs) were constitutively present and active in cholangiocytes, IL-6 increased p44/p42, but not p38 MAPK activity. PD098059 inhibited activation of p44/p42 MAPK in cholangiocytes and completely blocked DNA synthesis in response to IL-6 or LPS. These studies identify a critical role for the p44/p42 MAPK in cholangiocyte proliferation and demonstrate that the proliferative response of cholangiocytes to inflammatory mediators such as LPS involves IL-6-mediated activation of the p44/p42 MAPK pathway.