Objective:To determine whether HIV-infected patients have a deficiency of intracellular glutathione (GSH) in peripheral blood mononuclear cells (PBMC) and erythrocytes. Design:Initial experiments determining the stability of intracellular GSH preceded the measurement of GSH levels in 33 HIV-positive patients and 40 control subjects within 1 h of isolation of their blood cells. In addition, the susceptibility of erythrocytes to dapsone hydroxylamine-induced methaemoglobinaemia was evaluated. Methods:GSH levels were determined by an high-performance liquid chromatography method utilizing a fluorescent probe, monobromobimane. The bimane–GSH adduct formed in PBMC was also characterized by mass spectrometry. Methaemoglobin formation on exposure to dapsone hydroxylamine was determined spectrophotometrically. Results:GSH levels remained stable for only 1 h after cell isolation, thereafter showing a decrease of 20 and 60% at 4 and 24 h, respectively. There was no difference in the GSH levels in PBMC and erythrocytes of the HIV-positive patients compared with controls. The GSH levels were not related to the disease stage or to CD4+ cell counts. There was no difference in GSH levels in PBMC taken from trimethoprim–sulphamethoxazole-hypersensitive and non-hypersensitive patients. Methaemoglobinaemia on exposure of erythrocytes to dapsone hydroxylamine was concentration-dependent, but there was no significant difference between patients and controls. Conclusion:In contrast to previous studies, no deficiency of intracellular GSH in the PBMC and erythrocytes of HIV-infected patients was found. The discrepancy between studies may be methodological reflecting the instability of GSH, which requires prompt sample analysis.