The secretion of very-low-density lipoprotein from hepatocytes proceeds through the microtubules. In this study, the role of glutathione in the maintenance of intact microtubules and the secretion of very-low-density lipoprotein has been investigated. When rat hepatocytes were incubated with reagents that deplete glutathione (e.g., diethylmaleate, alpha-bromoisovalerylurea or allyl alcohol) or reacted directly with protein thiols (disulfiram), the secretion of very-low-density lipoprotein by the cells was inhibited and the microtubules were severely damaged as visualized by immunofluorescence staining. Both events occurred within 30 min; long before, an effect on the energy status of the cells became evident. The observed inhibition of the secretion therefore seems due to an effect of the toxicants on the microtubules. For alpha-bromoisovalerylurea, diethylmaleic acid and allyl alcohol, it may be related to glutathione depletion; preincubation of the hepatocytes with N-acetyl-L-cysteine reduced the decrease of glutathione by alpha-bromoisovalerylurea and allyl alcohol (but not of diethylmaleic acid) and almost completely prevented the inhibition of very-low-density lipoprotein secretion and microtubule damage. Depletion of glutathione may result in modification of a small group of essential free protein thiols. Disulfiram did not deplete glutathione, and N-acetyl-L-cysteine could not prevent the effects of disulfiram on microtubules. The binding to protein thiols of radiolabeled disulfiram, which binds to microtubules in vitro, was determined. At 0.2 mmol/L disulfiram, only 3% of total cellular protein thiols were conjugated, but secretion of very-low-density lipoprotein was already inhibited by 25%, and microtubules were severely affected. We propose that modification of a small fraction of cellular protein thiols results in the loss of microtubular ultrastructure and thereby leads to inhibition of very-low-density lipoprotein secretion.