Recent studies indicate that biliary lipids influence in vivo gallbladder absorption and solute-coupled water flow. To clarify the electrophysiologic effects that underlie this phenomenon, prairie dog gallbladders were mounted in an Ussing-type chamber, and the influence of bile acids and varying ratios of bile acids and biliary phospholipids on transepithelial potential difference (Vms), resistance (Rt), and short-circuit current (Isc) was examined. Exposure to 5 mmol/L taurodeoxycholate (TDC) resulted in inhibition of Vms (p less than 0.01) and Isc (p less than 0.01) and an increase (p less than 0.05) in Rt. Subsequent perfusion with bile acids and phospholipids (5 mmol/L TDC + 0.3 mmol/L phosphatidylcholine [PC]) led to continued inhibition of ion transport. In contrast, exposure to 5 mmol/L TDC + 1.7 mmol/L PC resulted in a significant increase in transport, as manifested by an increase in Vms (p less than 0.02) and Isc (p less than 0.01) and a decrease in Rt (p less than 0.05) compared with bile acids. These results indicate that the ratio of phospholipids to bile salts modulates ion transport across prairie dog gallbladder and that this ratio may be an important determinant of gallbladder absorption in health and disease.