It has been established that the cytoskeleton plays an important role in the secretory function of hepatocytes. We studied the effect of chlorpromazine (CPZ) and low calcium (LC) on the cytoskeleton of primary cultured hepatocytes using double-labelling immunofluorescence and secretion of fluorescein diacetate (FD) into the bile canaliculi (BC). The hepatocytes were obtained from 14-day-old male rats. They were cultured for 24 h in serum-free William's E medium with insulin and dexamethasone added to induce differentiation including bile canaliculus formation. After incubation with CPZ (200 μM) for 1 h, the BC became dilated and distorted and formed diverticula. Actin filaments around the BC became more prominent and the stress fibers decreased. CPZ did not affect the microtubules or cytokeratin filaments. Exposure to LC (20 μM) for 24 h caused a slight dilatation of the BC. Actin spread out over the cell periphery and appeared non-filamentous. Actin filaments around the BC appeared unchanged and the stress fibers disappeared. Microtubules and cytokeratin filaments were unaffected. Secretion of FD into the BC occurred normally after treatment with CPZ or LC. These results support the idea that the integrity of actin is not necessary for secretory function and that microtubules and intermediate filaments play a role in this process. The dilatation and diverticular formation in the BC induced by CPZ treatment suggests that a cytochalasin-like loss of contraction of the BC may explain the CPZ-induced decrease in bile flow observed in vivo.