The transcytotic vesicular pathway in isolated rat hepatocyte couplets was investigated using horseradish peroxidase. Ten to 20 min after horseradish peroxidase labeling, vesicles and tubules containing horseradish peroxidase were observed to be predominantly around the bile canaliculi. In hepatocytes incubated in a 4 degrees C medium for 10 min after horseradish peroxidase labeling, few horseradish peroxidase-containing structures were observed around the bile canaliculi, and the fine reticular immunofluorescence of microtubules was reduced. Cells treated with cytochalasin B (a microfilament inhibitor) showed a fair number of horseradish peroxidase-containing structures around the markedly dilated bile canaliculi and the distribution of microtubules was preserved. Cells labeled by horseradish peroxidase and then incubated for 10 min in a horseradish peroxidase-free medium containing 50 mumol/L of taurocholic acid, ursodeoxycholic acid or tauroursodeoxycholic acid had more tubular structures containing horseradish peroxidase around the bile canaliculi than control cells, whereas 50 mumol/L of taurochenodeoxycholic acid, taurodeoxycholic acid, dehydrocholic acid and taurodehydrocholic acid each failed to increase the number of tubular structures. These findings show that horseradish peroxidase was transported in hepatocyte couplets from the cell periphery to the bile canalicular front through the tubulovesicular pathway, depending on cytoplasmic microtubules. Cytoplasmic microfilaments appeared to play a minor role in this transport. Several specific bile acids such as taurocholic acid, ursodeoxycholic acid and tauroursodeoxycholic acid each promoted the tubular transformation.