Rhodamine 123 has been shown to be a substrate for P-glycoprotein in multidrug resistant cells. In the present investigation the disposition of rhodamine 123 was studied in the isolated perfused rat kidney. After exposing the kidneys to perfusate concentrations ranging from 10 to 1000 ng/ml, the renal clearance was 4-1 times the clearance by glomerular filtration, respectively, indicating active and saturable secretion of rhodamine 123. The rate-limiting step in secretion was found to be membrane passage from cell to tubular lumen. Suprisingly, renal clearance was not influenced by the P-glycoprotein inhibitors cyclosporin A or digoxin. However, pretreatment of the kidneys with verapamil and quinidine (inhibitors of both P-glycoprotein and organic cation transport) or cimetidine (organic cation transport inhibitor) resulted in a significantly reduced rhodamine 123 clearance, indicating that the renal organic cation carrier may be involved in active secretion. Rhodamine 123 accumulated extensively in the isolated perfused rat kidney; tissue concentrations of 270-360 times the perfusate concentration were determined. Similar accumulation ratios at different perfusate concentrations were found, suggesting that the compound enters the tubular cells by (facilitated) diffusion. In conclusion, rhodamine 123 accumulated extensively in the isolated perfused rat kidney and active renal secretion appears to be preferentially mediated by the organic cation carrier and not by P-glycoprotein.