Nonsteroidal anti-inflammatory drugs (NSAIDs) are antitumorigenic in humans as well as in animal models of intestinal neoplasia, such as the adenomatous polyposis coli ^Min/+ (Apc^Min/+) mouse. NSAIDs inhibit cyclooxygenase (COX) isozymes, which are responsible for the committed step in prostaglandin biosynthesis, and this has been considered the primary mechanism by which NSAIDs exert their antitumorigenic effects. However, mounting evidence suggests the existence of COX-independent mechanisms. In the present study, we attempted to clarify this issue by treating Apc^Min/+ mice bearing established tumors with NSAIDs (piroxicam and sulindac, 0.5 and 0.6 mg/mouse/day, respectively) for 6 days and concomitantly bypassing COX inhibition by treatment with the E prostaglandin (EP) receptor agonists 16,16-dimethyl-prostaglandin E₂ (PGE₂) and 17-phenyl-trinor-PGE₂ (10 μg each, three times daily) administered via gavage and/or i.p. routes. Treatment with piroxicam and sulindac resulted in 95% and 52% fewer tumors, respectively, and a higher ratio of apoptosis:mitosis in tumors from sulindac-treated mice as compared with controls. These effects were attenuated by concomitant EP receptor agonist treatment, suggesting PGE₂ is important in the maintenance of tumor integrity. Immunological sequestration of PGE₂ with an anti-PGE₂ monoclonal antibody likewise resulted in 33% fewer tumors in Apc^Min/+ mice relative to untreated controls, additionally substantiating a role for PGE₂ in tumorigenesis. The EP receptor subtype EP1 mediates the effects of PGE₂ by increasing intracellular calcium levels ([Ca²⁺]_i), whereas antagonism of EP1 has been shown to attenuate tumorigenesis in Apc^Min/+ mice. We demonstrate that [Ca²⁺]_i is significantly elevated in tumors of Apc^Min/+ mice relative to the adjacent normal-appearing mucosa. Furthermore, treatment with piroxicam results in significantly lower [Ca²⁺]_i in tumors, and this effect is attenuated by concomitant treatment with the EP1/EP3 receptor agonist 17-phenyl-trinor-PGE₂. Overall, our results suggest that NSAIDs exert their antitumorigenic effects, in part, via interference with PGE₂ biosynthesis, and these effects may be mediated through changes in intracellular calcium levels.