Hydroperoxides are major products of the reactions of radicals and singlet oxygen with amino acids, peptides, and proteins. These species can generate radicals in the presence of metal ions and oxidize thiols via nonradical reactions, but the effects of these materials on cells are poorly understood. In this study the exposure of murine macrophage-like cells to preformed peptide or protein hydroperoxides is shown to result in hydroperoxide consumption and cellular thiol oxidation; these effects precede loss of cell viability. N-acetyltryptophan methyl ester hydroperoxides, but not the decomposed species, decreased total cellular thiols and GSH, with the latter occurring more rapidly. Time-dependent inhibition of lysosomal cathepsins B and L was also observed, together with diminished caspase 3/7 activity. A number of other cytosolic thiol- and non-thiol-dependent enzymes were not affected significantly. Hydroperoxides formed on BSA did not deplete total thiols or GSH within cells, although such reactions are rapid in model systems. In contrast, selective inhibition of cathepsins B and L (but not cathepsin D or arylsulfatase) of the endosomal-lysosomal system was detected, consistent with localization within these compartments. Decomposed BSA hydroperoxides did not induce these effects, indicating a requirement for the hydroperoxide group. The differences between these hydroperoxides are ascribed to their mechanisms of penetration into cells. Overall these studies provide valuable data on the initial cellular events arising from exposure to exogenous protein and amino acid peroxides and indicate that cellular thiols are a major target. This selective oxidation may modulate cellular redox balance and subsequent cell behavior.