Chronic cholestasis is characteristic of many human liver diseases. Renal injury has been often associated with this type of disease. The aim of this study was to evaluate the effect of cholestasis on kidney mitochondrial bioenergetics following in vivo chronic administration of alpha-naphthyl-isothiocyanate (ANIT), a known cholestatic agent. Serum markers of renal injury, kidney morphology and endogenous adenine nucleotides were measured in ANIT-treated rats (80 mg/kg per week s.c. for 16 weeks). Changes in membrane potential and mitochondrial respiration as well as alterations in mitochondrial calcium homeostasis were monitored. Cholestatic animals shown no alterations in renal morphology when compared with control. Additionally, following chronic ANIT administration, no significant alterations in mitochondrial respiratory function have been shown. The phosphorylation capacity of cholestatic kidney mitochondria was enhanced. Associated with these parameters, mitochondria from treated animals exhibited a decreased susceptibility to disruption of mitochondrial calcium homeostasis, due to permeability transition induction. These data suggest that, despite being submitted to chronic treatment with ANIT, kidney mitochondria from cholestasis-induced rats present some defense mechanisms to circumvent this aggression. They show improved phosphorylative capacity and, moreover, a decreased susceptibility to mitochondrial permeability transition induction, probably due to adaptative mechanisms of calcium transport.