Changes in the concentration of intracellular Ca2+ may be an important component of the mechanism of adriamycin toxicity to tumor cells. Adriamycin interacts with the plasma membrane, a phenomenon which may lead to a direct modulation of Ca2+ transport proteins or, since the drug is a quinone, may lead to indirect changes in Ca2+ homeostasis induced by oxidative stress to the cell. The calcium content of L1210 murine leukemia cells treated with adriamycin for up to 6 hr was estimated using the cell-impermeant dye arsenazo-III. Pools of intracellular Ca2+ were released to the extracellular compartment, where they reacted with the dye by sequential treatment of the cells with m-fluorocarbonylcyanidediphenylhydrazone (FCCP) and the Ca2+-ionophore A23187. Pretreatment of L1210 cells with ruthenium red (5 microM) selectively decreased the FCCP-releasable Ca2+ pool, which suggested it was mitochondrial in origin. Continuous exposure of L1210 murine leukemia cells in vitro to 5 or 10 microM adriamycin for 2 hr did not produce any change in the intracellular concentration of releasable Ca2+; at 4 hr, however, the total releasable pool of Ca2+ rose by 29% and 46% for 5 and 10 microM adriamycin respectively. This increase was seen predominantly in the mitochondrial pool. Exposure of L1210 cells to the quinone, menadione, also increased the releasable pools of cellular Ca2+ but like adriamycin, only after an incubation period of 4 hr. These results contrasted with a rapid decrease in mitochondrial Ca2+ concentration produced by a short (5 min) exposure to 500 microM t-butylhydroperoxide, a generator of free radicals. After treatment with 8 mM lidocaine, a membrane fluidizing agent, there was a rapid fall in extramitochondrial Ca2+. These findings suggest that changes in L1210 Ca2+ homeostasis induced by adriamycin and menadione are late, and possibly common, events of quinone toxicity to L1210 cells, adriamycin does not have an immediate effect on Ca2+ ion transport produced by the direct interaction of the antibiotic with the plasma membrane, and oxidative stress induced by redox-active quinones may not be important for the induction of toxicity in neoplastic cells.