Ochratoxin A (OA) and oosporein (OSN) are two mycotoxins that may cause nephrotoxicity through either mitochondrial dysfunction or lipid peroxidation. Using isolated rat renal proximal tubules in suspension, the cellular events preceding OA- or OSN-induced cytotoxicity were investigated. OA and OSN decreased tubule viability in a concentration (0–1 m m)- and time (0–4 hr)-dependent manner, with initial decreases occurring 1 hr after exposure. Tubule basal and nystatin-stimulated oxygen consumption decreased before cell death after OA (0.5 and 1 m m) and 0.25 m m t-butyl hydroperoxide (TBHP) exposure, but did not decrease after OSN exposure (0.25–1 m m). The oxidant TBHP was used as a positive control in these studies. Direct probing of mitochondrial function within proximal tubules confirmed the toxicity of OA to mitochondria. Respiration was reduced in the absence and presence of a phosphate acceptor using site I (glutamate/malate) and site II (succinate) respiratory substrates 15 and 30 min after exposure to 1 m m OA. Lipid peroxidation preceded cell death after exposure to 1 m m OA and 0.25 m m TBHP, but did not occur after exposure to 1 m m OSN. Deferoxamine (1 m m) pretreatment before the addition of 1 m m OA or OSN prevented OA-induced lipid peroxidation, but did not prevent OA- or OSN-induced cytotoxicity. In contrast, deferoxamine pretreatment prevented lipid peroxidation, mitochondrial dysfunction, and the loss of tubule viability after exposure to 0.25 m m TBHP. This study shows that mitochondrial dysfunction is an early event during the development of OA toxicity, but not in OSN-induced toxicity. Furthermore, iron-mediated lipid peroxidation does not contribute to OA- or OSN-induced proximal tubule cell death.