The mechanism for renal tubular secretion of digoxin as well as its interaction with quinidine or verapamil were investigated using the isolated perfused rat kidney. [3H]Digoxin was instantaneously administered into the renal artery together with [14C]inulin and Evans blue-albumin, and renal venous and urinary outflow curves were measured. The ratio of fractional excretion to filtration fraction for digoxin was 2.40 +/- 0.40, indicating involvement of tubular secretion. Quinidine and verapamil decreased the ratio of fractional excretion to filtration fraction in a concentration-dependent manner, and this inhibition was indicated to occur at transport from cells to lumen across luminal membranes. Neither tetraethylammonium nor p-aminohippurate affected the renal handling of digoxin. Because ouabain and digitoxose showed no influence on the value of fractional excretion to filtration fractions, Na+,K(+)-ATPase is not involved in the tubular secretion of digoxin. A metabolic inhibitor, 2,4-dinitrophenol, markedly inhibited digoxin secretion. Agents that bind to P-glycoprotein, such as vinblastine, daunorubicin and reserpine, markedly inhibited the secretion of digoxin. Recently, we have found that digoxin is a substrate transported by P-glycoprotein. The findings obtained here support the hypothesis that digoxin is secreted by P-glycoprotein located on the luminal membrane of renal tubular epithelial cells, and that clinically important interactions with quinidine and verapamil are caused by the inhibition of P-glycoprotein.