Summary Reduced glutathione (GSH) has been demonstrated in benign and malignant human breast lesions using a newly.developed histofluorescence technique. GSH was present in every lesion and in each case was localised to the epithelium. A semi-quantitative assessment revealed a moderate amount of GSH in normal epithelium and fibroadenoma and a high level in apocrine metaplasia, epitheliosis and intraduct carcinoma. Invasive ductal carcinoma contained a variable amount of GSH. Correlation between fluorescence intensity and histological grade of ductal carcinomas was almost statistically significant but a relationship to oestrogen receptor status was not detected. The rapid assessment of GSH in breast cancer may aid in the selection of optimum chemotherapeutic regimens.Reduced glutathione (GSH) is a thiol containing tripeptide which is involved in a variety of cellular functions. GSH has a role in the detoxification of drugs and carcinogens, protection of cells from free radical damage and reactive oxygen compounds, participates in protein and DNA synthesis and in the regulation of enzyme activity (Meister & Anderson, 1983;Orrenius & Moldeus, 1984;Chasseaud, 1979). Altered levels of GSH are frequently observed in experimental animal neoplasia (Roomi et al., 1985, Fiala et al., 1976 but GSH levels in human neoplasia have been reported only for carcinomas of the stomach and colon (Siegers et al., 1983;Siegers et al., 1984). GSH in human breast tissue has not been investigated previously, although GSH-associated enzymes have been studied both biochemically (Di Ilio et al., 1985) and histochemically (Levine et al., 1983;Bard et al., 1986).Tumour GSH appears to be an important factor in determining the effectiveness of a variety of anti-cancer chemotherapeutic drugs, in general a low level of GSH being associated with increased chemotherapeutic efficiency (Arrick & Nathan, 1984;Romine and Kessel, 1986;Crook et al., 1986).We have studied the localisation and amount of GSH in normal and pathological human breast tissue using our recently developed specific histofluorescence method for GSH (Murray et al., 1986 (dichroic mirror) for epi-illumination (set at position 1, 50% transmission at 400 nm), a UGI excitation filter (band pass filter, peak transmission 365nm) and a K430 high pass emission filter (50% transmission at 430 nm).The distribution of fluorescence in each section was recorded and the intensity of GSH fluorescence assessed in semi-quantitatively on a 4-point scale (0=no fluorescence, + = slight fluorescence, + + =moderate fluorescence, + + + = bright fluorescence).Relationship between GSH concentration and histofluorescence Five known concentrations of authentic GSH (2, 4, 6, 8, 10mM) in 0.1 M formic acid were each mixed with 1% polyvinylpyrrolidone (PVP: 2 ml GSH solution plus 1 ml PVP), droplets applied to glass slides and then dried in air. The slides were then reacted with o-phthalaldehyde as described above, examined with fluorescence microscopy and the fluorescence intensity assessed semi-quantitati...