The biliary output of bile acids and lipids is tightly coupled. The ability of the natural bile acid glycocholate to trigger biliary lipid secretion was compared with that of the fluorescent bile acid analogue cholyl-lysylfluorescein (cholyl-lys-F). When administered as a 5 min pulse of 2.5 mumol/min to bile acid-depleted rat livers perfused under recycling conditions, glycocholate produced well-defined peaks of phospholipid and cholesterol output, and of bile flow, which were coincident with the peak of bile acid output. Although cholyl-lys-F did trigger biliary lipid secretion, its time course of appearance was delayed and well-defined peaks of output were not observed. However, the increased biliary output of phospholipid and cholesterol was coincident with that of bile acids and, as judged by phospholipid/bile acid and cholesterol/bile acid ratios, cholyl-lys-F was as effective as glycocholate in triggering biliary lipid output. When administered to livers perfused under single pass conditions, perfusate to bile transfer of glycocholate was > 85% at infusion rates of up to 5 mumol/min whereas transfer of cholyl-lys-F showed saturation at infusion rates of > 0.2 mumol/min; the time course of biliary output of both bile acids was similar. Thus, under recycling conditions, cholyl-lys-F not taken up during first pass will be continually represented for transfer to bile, explaining why bile acid and lipid output did not occur as well-defined peaks.