Gliotoxin is an epidithiodioxopiperazine compound which can both react with sulfhydryl groups and form hydrogen peroxide. Rat liver mitochondria contain a prooxidant-regulated specific Ca2+ release pathway. Here we report that gliotoxin at low concentrations stimulates Ca2+ release via this pathway in isolated mitochondria. Ca2+ release is not promoted by gliotoxin exposed to disulfide-reducing reagents prior to addition to mitochondria or when its disulfide moiety is dimethylated. Gliotoxin is equally effective in glutathione-depleted and glutathione-adequate mitochondria. This and the unchanged mitochondrial oxygen consumption in the presence of gliotoxin suggest that the compound stimulates Ca2+ release by reacting with critical mitochondrial thiol compounds and not by increasing hydrogen peroxide formation in mitochondria. The gliotoxin-induced Ca2+ release is paralleled by hydrolysis of mitochondrial pyridine nucleotides, and both pyridine nucleotide hydrolysis and Ca2+ release are inhibited by cyclosporin A. These findings provide further insight into the regulation of Ca2+ release from intact mitochondria.