The present study evaluates the effect of free radicals generated by xanthine oxidase-catalyzed oxidation of hypoxanthine on cellular function of isolated rat pancreatic acinar cells. The results show that a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+]i) preceded all other morphological and functional alterations investigated. Radical-induced [Ca2+]i increase was largely inhibited by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, which prevents Ca2+ release from intracellular stores, but not by Ca2(+)-depleted medium. Radicals released Ca2+ from thapsigargin-insensitive, ryanodine-sensitive intracellular stores, whereas the secretagogue caerulein at physiological concentrations mainly released Ca2+ from thapsigargin-sensitive stores. In contrast to effects of the secretagogue, radical-induced Ca2+ changes did not cause luminal protein secretion but cell death. In single-cell measurements, both secretagogue and radicals induced oscillations of [Ca2+]i. Radical-induced oscillations had a lower frequency but similar amplitude when compared with caerulein-induced oscillations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and ryanodine, which prevented the radical-induced Ca2+ increase without altering the generation of radicals, markedly reduced the radical-induced cell damage. These results suggest that the Ca2+ increase mediates the radical-induced cell injury. The studies also indicate that not only the extent and duration but also the origin of [Ca2+]i release as well as the frequency of Ca2+ oscillations may determine whether a pancreatic acinar cell will secrete or die.