Relatively similar concentrations of the inorganic ions were detected in rat, rabbit, and dog bile; however, dog bile had a higher concentration of protein, cholesterol, phospholipid phosphorous, and percentage solids than rat bile, and rabbit bile had the lowest concentration. The biliary excretion of bile acids was altered in each species by: (1) interruption of the enterohepatic circulation; (2) rapid administration of an exogenous load of bile acids; and (3) constant infusion of an exogenous load of bile acids. Bile acid and phospholipid phosphorous concentration and percentage solids increased after bile acid administration in all three species; however, species differences in bilirubin concentration were observed and a marked decrease was detected in rabbit and dog bile but it markedly increased in rat bile. When the enterohepatic circulation was interrupted in the dog and rat, the bile acid concentration markedly decreased with only minor changes in bile flow. This not only supports the theory that there is a bile salt independent fraction of bile formation, but also demonstrates that canalicular bile formation can be maintained at relatively normal rates with almost no excretion of bile acids. Marked discrepancy between bile acid excretion and bile flow was observed in the rat after bile acid administration, in that a marked increase in bile acid excretion was observed but little or no increase in flow. When bile flow was plotted against bile acid excretion for the three species, the slope of the line was less during bile acid administration than during depletion, indicating that the bile acids are accompanied by less water during bile acid administration than during depletion. Variation in the bile flow intercept with zero bile acid excretion (thought to represent the bile salt-independent fraction) was relatively large, which is probably due in part to alteration in the production of the bile salt independent fraction when bile acid secretion is altered. It appears that both the choleretic property of bile acids varies during various rates of bile acid excretion and the bile salt-independent fraction is not constant. Therefore, calculation of the bile salt independent fraction as previously performed should be interpreted with extreme caution. Thus, it appears difficult to determine the quantitative importance of bile acid excretion in bile formation.