Recently, we have reported that 3,5-dialkyl substitution of paracetamol, in contrast to 3-monoalkyl substitution, prevented the paracetamol-induced toxicity in freshly isolated rat hepatocytes without having any effect on its cytochrome P-450 mediated bioactivation to reactive N-acetyl-p-benzoquinone imines (NAPQI). In the present study the mechanism of this prevention of toxicity, with special emphasis on oxidative stress, was studied in more detail in freshly isolated rat hepatocytes, using paracetamol, 3-methyl-, 3,5-dimethyl-paracetamol, synthetic NAPQI and 3,5-dimethyl-NAPQI. 3-Methyl-paracetamol was found to induce glutathione (GSH) depletion, lipid-peroxidation and cytotoxicity in hepatocytes to the same extent as paracetamol. 3,5-Dimethyl-paracetamol, however, even when added in a ten-fold higher concentration when compared to paracetamol, did not induce any of these effects. Similar differences of toxicity were observed between NAPQI and 3,5-dimethyl-NAPQI; 3,5-dimethyl-NAPQI, in contrast to NAPQI, did not reduce protein thiol levels, did not induce GSH depletion, lipid-peroxidation nor cytotoxicity. Only after artificial depletion of GSH levels in the hepatocytes by DEM or BCNU, 3,5-dimethyl-NAPQI was cytotoxic. This effect was accompanied by depletion of protein thiol levels, but not by lipid-peroxidation. Addition of the disulfide reducing agent, dithiothreitol, prevented the artificially created cytotoxicity of 3,5-dimethyl-NAPQI. It is concluded that prevention of paracetamol-induced toxicity by 3,5-dialkyl substitution is primarily due to prevention of irreversible GSH-depletion, presumably caused by the inability of 3,5-dialkyl-NAPQI to conjugate with thiols. As a result, the GSH-dependent cellular defense mechanism against potential oxidative cellular injury by 3,5-dialkyl-NAPQI is left unimpaired. Our observations indicate that a compound, not capable of covalent binding to thiol groups of proteins, can induce toxicity solely as a result of protein thiol oxidation without inducing lipid-peroxidation.