Glutathione appears to be a major osmotic factor in the generation of bile salt-independent flow (BSIF). This study was designed to investigate its importance in the pathology of 17-alpha-ethinyl estradiol (EE)-induced cholestasis. Five-day EE treatment at the dose level of 5 mg/kg/day significantly decreased bile flow (57% of controls) and biliary glutathione secretion. Evaluation of the contribution of bile salt dependent flow (BSDF), glutathione dependent flow (GSDF) and the bile flow generated independently of both bile salts and glutathione (BS-GSIF) revealed that EE decreased all portions of the flow (63, 44 and 52% of control values, respectively). At 4 and 20 h after a single administration of the same EE dose, a significant diminution of bile flow was noted (decreases of 17 and 29%, respectively) in association with a significant fall in biliary glutathione content. Under these conditions, BSDF and BS-GSIF were not modified (98 and 112% of control BSDF values, respectively; 96 and 99% of control BS-GSIF values, respectively) while GSDF was decreased markedly, representing 65 and 50% of control values. Biliary glutathione secretion was diminished without modification of liver and blood glutathione concentration or redox status following single EE dose whereas, after 5 days of EE treatment, a significant increase in liver glutathione was observed, suggesting that EE may interfere with the glutathione secretory process. This study demonstrates that EE rapidly alters biliary glutathione content, leading to a marked decline in GSDF. This reduction may explain the decrease in BSIF produced by EE at the outset of cholestasis.