The bile canaliculi of hepatocytes contract spontaneously, and it is hypothesized that this canalicular motility provides a propulsive force for normal intrahepatic bile flow. Cytochalasin disrupts actin polymerization, inhibits contraction, and decreases bile flow. We investigated whether this cholestasis was associated with impaired canalicular secretion. Isolated rat hepatocyte doublets, with and without incubation with 2 mumol/L cytochalasin D (cytD), were superfused, under first-order conditions, to steady state with fluorescein isothiocyanate-labeled glycocholic acid (FITC-GC) and carboxy-4′,5′-dimethylfluorescein diacetate (CMFD), which are fluorescent substrates for the bile acid and the nonbile acid organic anion transport pathways, respectively. Fluorescent microscopic images were quantified and the data analyzed by noncompartmental and compartmental kinetic methods. cytD dilated the canalicular spaces fivefold but did not change the proportion of doublets that secreted either probe. Cytochalasin did not affect the mean cellular transit times of FITC-GC (2.8 and 2.5 minutes for control and cytochalasin-treated groups, respectively) and of carboxy-4′,5′-dimethylfluorescein (3.8 and 3.7 minutes, respectively). Analysis with a three-compartment model gave estimates of the rate constants for canalicular secretion: 0.21 ± 0.04 and 0.22 ± 0.03 min−1 in control and treated cells, respectively, for FITC-GC, and 0.14 ± 0.01 and 0.16 ± 0.02 min−1, respectively, for carboxy-dimethylfluorescein. When kinetics are first-order, the canalicular secretion of organic anions is not altered by actin disruptive agents, suggesting that actin filaments do not modulate the function or distribution of these transporters. This suggests that impaired contractility rather than impaired canalicular secretion is the mechanism of cytD-induced cholestasis.