Using isolated liver mitochondria we show that low concentrations of TBT (0.5 muM) cause the release of mitochondrial cytochrome c, in the presence of Ca2+. This is reflected in a rapid loss of membrane potential (Deltapsi(m)), and a large-amplitude swelling characteristic of mitochondrial permeability transition (MPT). Despite this, the inclusion of cyclosporin A could not prevent the release of cytochrome c. Further, in the absence of Ca2+, low concentrations of TBT (0.5 muM) resulted in a slow sub-maximal shift of Deltapsi(m), not characteristic of MPT, which was still paralleled by a release of cytochrome c. Further experiments showed that the loss of Deltapsi(m) in the absence of Ca2+ was due to a combination of inhibition of respiration and a direct uncoupling effect on the respiratory chain. Under these conditions, rapid swelling of mitochondria could be demonstrated, due to chloride exchange over the inner mitochondrial membrane. Taken together these data suggest that TBT can induce the release of cytochrome c in intact cells by at least two mechanisms. The first and critical mechanism is initiated immediately the mitochondria sense the presence of TBT and involves a slow loss of Deltapsi(m) and induction of swelling, which allows release of cytochrome c in a relatively non-specific manner and independently from a rise in [Ca2+](i). The second mechanism involves the induction of formal MPT as intracellular [Ca2+](i) increases. These data help to explain previous observations in intact lymphocytes demonstrating TBT-induced release of mitochondrial cytochrome c in the absence of a rise in [Ca2+], (Stridh, H., Gigliotti, D., Orrenius, S., and Cotgreave, I. A. (1999) Biochem. Biophys. Res. Commun. 266, 460-465). (C) 2002 Elsevier Science (USA).