Objective: To identify proteins of the bile canalicular membrane (BCM) of rat liver involved in canalicular bile secretion. Experimental Design: The initial step will be to subfractionate the BCM rich preparaton (continuous sucrose gradient) and to analyze the components of each fraction (enzymes, protein electrophoresis, electron microscopy). Proteins will be solubilized from fractions by Lubrol WX, Triton X-100 and 0,9% NaCl and antibodies developed against specific proteins to determine their interacellular localization by electron microscopy (antibodies incubated with liver slices followed by incubation with peroxidase-anti rabbit IgG complex). The kinetics of binding of membrane subfractions to Na22 and to labeled bile salts will be determined by incubations followed by millipore filtration. The kinetics of binding of these solutes to solubilized proteins will be determined following the rate of dialysis of labeled substrates from a double dialysis chamber. Turnover rate of the proteins involved will be determined following their decay after C14 guanidino-arginine labeling. Finally, the effect of ouabain, scillaren, estrogens, tauvo 24,25 dihydrofusidate and phenobarbital, substances which modify bile secretion, on the turnover and binding affinities of these membrane proteins will be assessed.