Anionic polypeptide fraction (APF) is a phospholipid- and calcium-binding apoprotein present in animal and human bile, predominantly associated with cholesterol-phospholipid vesicles. In bile, the protein may play a physiological role in preventing precipitation of calcium salts. APF has also been suggested to be of regulatory importance in the process of biliary lipid secretion. The aim of the present study was to investigate whether the secretion rates of APF and that of biliary lipids are coupled, which would support a physiological role of APF in biliary lipid secretion. Biliary secretion rates of bile acids, phospholipids, and cholesterol were experimentally modulated in three different rat models. Secretion rates of APF were compared with that of bile acids, lipids, and with that of two other biliary proteins, the lysosomal protein β-glucuronidase and apolipoprotein A-I (apo A-I). Model 1: diurnal variation in bile formation during chronic bile diversion; model 2: specific inhibition of biliary phospholipid and cholesterol, but not of bile acid secretion by infusion of the organic anion, sulfated lithocholyltaurine; model 3: acute interruption of the enterohepatic circulation in unanesthetized rats. The diurnal variation in bile formation involved a parallel increase of the biliary secretion rates of bile acids (+56 ± 7%, mean ± SD), phospholipids (+53 ± 29%), cholesterol (+73 ± 54%), and APF (+72 ± 86%) during the night phase of the cycle. Infusion of sulfated lithocholyltaurine inhibited biliary phospholipid and cholesterol secretion (−78 ± 15%, and −54 ± 25%, respectively), but did not affect biliary bile acid or APF secretion rate (−19 ± 14%, and +12 ± 107%, respectively). Within 4 hours after interruption of the enterohepatic circulation, bile secretion rates for bile acids (−92 ± 3%), phospholipids (−74 ± 13%), cholesterol (−64 ± 8%), and APF (−58 ± 24%) rapidly declined to a new steady-state level. Correlation analysis using the data from the three experimental models indicated that the biliary secretion rate of APF was independent from that of phospholipids, cholesterol, β-glucuronidase, and, presumably, apolipoprotein A-I, and positively correlated to bile acid secretion rate and bile flow. The data from three experimental models indicate that the biliary secretion rates of APF and of phospholipids/cholesterol are not coupled and, therefore, do not support a direct physiological role of APF secretion in biliary lipid secretion. APF secretion into bile may, at least partially, be controlled by biliary bile acid secretion.