The pyrrolizidine alkaloid senecionine has been shown to produce an increase in cytosolic free Ca2+ concentration in isolated hepatocytes that correlated with an increase in cellular toxicity. The cytotoxicity was greater in the absence of extracellular Ca2+ than in its presence, suggesting that alterations in intracellular Ca2+ distribution, and not an influx of extracellular Ca2+, were responsible for the senecionine-induced hepatotoxicity. The effect of senecionine, as well as the effects of trans-4-OH-2-hexenal (t-4HH), a microsomal metabolite of senecionine, and a related alkenal, trans-2-hexenal, on the sequestration of Ca2+ in mitochondrial and extramitochondrial compartments were examined in isolated hepatocytes. Each of the test compounds elicited a decrease in the available extramitochondrial Ca2+ stores that was inhibited by pretreatment with the thiol group reducing agent, dithiothreitol. Senecionine and t-4HH decreased the level of Ca2+ sequestered in the mitochondrial compartment of hepatocytes. The presence of a pyridine nucleotide reducing agent, beta-hydroxybutyrate, inhibited this reduction. These results suggest that both senecionine and t-4HH inhibit the sequestration of Ca2+ in extramitochondrial and mitochondrial compartments possibly by inactivating free sulfhydryl groups and oxidizing pyridine nucleotides respectively.