Sprague-Dawley rats were treated with a combination of buthionine sulfoximine (2.5 mmol/kg body wt) and diethyl maleate (6.2 mmol/kg). After 4 h, the hepatic glutathione was depleted to 0.02 +/- 0.01 mumol/g liver (mean +/- SD, n = 6) (4.73 +/- 0.29 mumol/g in controls (n = 6), p < 0.005). The bile flow rate was lower in treated animals than in controls (39.0 +/- 11.2 vs. 73.7 +/- 13.7 microliters.kg-1.min-1, p < 0.005). Bile concentrations of bile acids, phospholipids, and cholesterol were not changed compared to controls, while glutathione was virtually absent from the bile. A bolus of indocyanine green (6.5 mumol/kg), rose bengal (6.5 mumol/kg), or sulfobromophthalein-glutathione conjugate (20 mumol/kg) was injected i.v. and the biliary excretion was monitored. In glutathione-depleted rats, the excretion of indocyanine green was delayed, and the cumulative excretion in 90 min was 36 +/- 8% (n = 6) of the injected dose (79 +/- 18% in controls (n = 7), p < 0.005). The bile concentration of indocyanine green was lower in the glutathione-depleted rats than in controls, while the other dyes increased. The plasma disappearance curve of indocyanine green was not significantly altered by the treatment, whereas the hepatic retention of indocyanine green 90 min after the injection was significantly increased (58 +/- 12% of the injected dose vs. 8 +/- 2% in controls, p < 0.005). The results indicate that depletion of hepatic glutathione inhibits biliary excretion of indocyanine green.