Cyclooxygenase-2 (Cox-2) mediates various inflammatory responses and is expressed in pancreatic tissue from patients with chronic pancreatitis. To examine the role of Cox-2 in chronic pancreatitis, we investigated its participation in regulating functions of pancreatic stellate cells (PSCs), using isolated rat PSCs. Cox-2 was expressed in culture-activated PSCs but not in freshly isolated quiescent PSCs. TGF-₁, interleukin (IL)-1, and IL-6 enhanced Cox-2 expression in activated PSCs, concomitantly increasing the expression of -smooth muscle actin (-SMA), a parameter of PSC activation. The Cox-2 inhibitor NS-398 blocked culture-activation of freshly isolated quiescent PSCs. NS-398 also inhibited the enhancement of -SMA expression by TGF-₁, IL-1 and IL-6 in activated PSCs. These data indiate that Cox-2 is required for the initiation and promotion of PSC activation. We further investigated the mechanism by which cytokines enhance Cox-2 expression in PSCs. Adenovirus-mediated expression of dominant-negative Smad2/3 inhibited the increase in expression of Cox-2, -SMA, and collagen-1 mediated by TGF-₁ in activated PSCs. Moreover, dominant-negative Smad2/3 expression attenuated the expression of Cox-2 and -SMA enhanced by IL-1 and IL-6. Anti-TGF- neutralizing antibody also attenuated the increase in Cox-2 and -SMA expression caused by IL-1 and IL-6. IL-6 as well as IL-1 enhanced TGF- secretion from PSCs. These data indicate that Smad2/3-dependent pathway plays a central role in Cox-2 induction by TGF-₁, IL-1, and IL-6. Furthermore, IL-1 and IL-6 promote PSC activation by enhancing Cox-2 expression indirectly through Smad2/3-dependent pathway via increasing TGF-₁ secretion from PSCs.