Indomethacin and other nonsteroidal anti-inflammatory drugs are commonly used to indirectly deduce the possible role of PGs in a process being studied. The objective of this study was to determine if indomethacin, at concentrations comparable to plasma and tissue levels obtained in humans taking therapeutic doses, predisposes human gastric cells to injury through inhibition of PGs or acts through an alternate mechanism. The role of intracellular Ca²⁺ in this damaging process was also assessed. Indomethacin pretreatment, although by itself nondamaging, was associated with elevated intracellular Ca²⁺ concentrations and an increased cellular permeability, an effect that was dependent on extracellular Ca²⁺. Furthermore, indomethacin pretreatment significantly predisposed AGS cells to injury induced by two dissimilar agents (deoxycholate and A-23187), both of which are associated with intracellular Ca²⁺ accumulation. The addition of exogenous PGs did not reverse the predisposition to injury induced by indomethacin. The observed effects of indomethacin were dependent on concentration and not on ability to inhibit PG synthesis. Similar effects were not observed with equipotent concentrations of ibuprofen or aspirin. Finally, the exacerbation of deoxycholate-induced injury induced by indomethacin was not observed when extracellular Ca²⁺ was removed. Indomethacin, by disturbing intracellular Ca²⁺ homeostasis, predisposes human gastric cells to injury through mechanisms independent of PG synthesis. The current study suggests that data resulting from studies employing only indomethacin as a PG synthesis inhibitor should be interpreted with caution.