The 14C-labeled, 35S-labeled, and unlabeled nephrotoxic cysteine conjugates S-(1,2-dichlorovinyl)-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)- L-cysteine, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine, S-(1,2,3,4,4-pentachlorobutadienyl)-L- cysteine (PCBC), and S-(1,1,2,3,3,3-hexafluoropropyl)-L-cysteine were synthesized and their toxicities were compared in isolated rat renal mitochondria. Inhibition of respiration, covalent binding to macromolecules, metabolism by mitochondria, metabolism by a purified cysteine conjugate beta-lyase (beta-lyase), and octanol/water partition coefficients were studied. All of the conjugates inhibited mitochondrial state 3 respiration. Only PCBC was found to uncouple oxidative phosphorylation. (Aminooxy)acetic acid, a beta-lyase inhibitor, blocked the effects of the conjugates on state 3 respiration except for the uncoupling effect of PCBC, which was not blocked. Binding of 35S label to macromolecules was observed after treatment with each of the 35S-labeled conjugates, and (aminooxy)acetic acid blocked the binding. The relative amounts of metabolism of the conjugates did not correlate well with their relative binding and toxicities, indicating some differential reactivity of metabolites and/or selectivity for binding targets. Some of the binding from 35S-labeled conjugates was removed by treatment with the disulfide-reducing agent dithiothreitol, suggesting that some of the binding was via mixed disulfides. The amount of dithiothreitol-sensitive binding differed among the conjugates. The metabolism of PCBC by permeabilized mitochondria, but not by a purified beta-lyase, was consistent with its relative toxicity and covalent binding, suggesting the involvement of other beta-lyase enzymes in the activation of PCBC to toxic species in mitochondria.