Physical-chemical and biological studies of hepatic bile suggest that biliary phospholipid molecules are secreted as unilamellar vesicles. Systematic ultrastructural studies of bile canaliculi were undertaken to visualize this event. Liver tissue was obtained from normal adult male rats (control), from bile salt-depleted rats (by overnight biliary diversion), and from depleted rats infused intravenously with a hydrophilic-hydrophobic congener series of common taurine-conjugated bile salts. Livers were fixed in situ either by modified chemical methods or by ultrarapid cryofixation. In control rats, chemical fixation revealed unilamellar vesicles 63 ± 17 (± SD) nm in diameter, mostly free within canalicular lumena. Vesicles were infrequent in canaliculi of bile salt-depleted rats, but were present in canaliculi of rats infused with taurocholate. In cryofixed liver tissue, vesicles 67 ± 13 nm in diameter were observed in canaliculi of control rats and bile-salt depleted rats infused with common bile salts. The majority of these vesicles were affixed to the luminal side of the canalicular membrane. The average number of vesicles per bile canaliculus was in agreement with that estimated on the basis of biliary phospholipid secretion rates, mean vesicle size, and area of close-packed phosphatidylcholine molecules. By immunoelectron microscopy, canalicular vesicles were free of actin and of a 100 kDa canalicular membrane protein. We conclude that biliary phospholipid molecules are secreted from hepatocytes into bile canalicular lumena as unilamellar vesicles ~63-67 nm in average diameter. We postulate that this secretion mechanism involves lumenal bile salt-induced vesiculation of lipid microdomains in the exoplasmic hemileaflet of the canalicular membrane.