The mechanisms underlying agonist-induced oscillations in intracellular free calcium ion concentration ([Ca2+]i) in hepatocytes were investigated by utilizing tert-butyl hydroperoxide (TBHP) as a tool to perturb hepatocyte Ca2+ homeostasis independent of receptor activation. In permeabilized hepatocytes, TBHP inhibited Ca2+ uptake into the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool and increased the sensitivity to InsP3 for Ca2+ release. The effects of TBHP could be mimicked by addition of oxidized glutathione (GSSG) and reversed by pretreatment with dithiothreitol. TBHP and GSSG had no effect on the metabolic degradation of [3H]InsP3 in permeabilized cells. The effect of TBHP on [Ca2+]i in intact cells was investigated by digital imaging fluorescence microscopy of Fura-2-loaded primary cultured hepatocytes. TBHP treatment initiated a series of [Ca2+]i oscillations similar to those caused by Ca2(+)-mobilizing hormones. Moreover, in common with the actions of hormones in these cells (Rooney, T.A., Sass, E., and Thomas, A,P. (1990) J. Biol. Chem. 265, 10792-10796), the [Ca2+]i oscillations induced by TBHP propagated through the cell as Ca2+ waves, originating from a discrete subcellular locus identical to that for phenylephrine-induced [Ca2+]i oscillations. The Ca2+ waves induced by TBHP had similar rates of progress (24-27 microns.s-1) to those generated by phenylephrine. Removal of extracellular Ca2+ increased the initial latency of the TBHP responses, but had no effect on the amplitude or rate of propagation of the Ca2+ waves. Addition of TBHP to cells in the presence of phenylephrine converted the oscillatory phenylephrine [Ca2+]i response into a sustained [Ca2+]i increase. The effects of TBHP in intact cells occurred in the absence of any stimulated inositol polyphosphate formation as measured in populations of [3H]inositol-labeled hepatocytes. The data indicate that spatially organized [Ca2+]i oscillations in intact hepatocytes can occur without any requirement for phospholipase C activation. Furthermore, for agents that act by mobilizing Ca2+ from the InsP3-sensitive pool, the kinetics of the Ca2+ release phase of the [Ca2+]i oscillations appears to be independent of the nature of the stimulus.