It has been suggested that modification of the pericanalicular microfilament network (F-actin) plays a role in cholestasis. The purposes of this study were to assess (i) the process of F-actin network reorganization in isolated rat hepatocyte couplets (IRHC) in order to define the optimal study conditions in this model, (ii) the effect of cholestatic and hepatotoxic but non-cholestatic compounds on F-actin distribution in IRHC. F-actin was stained with fluorescein isothiocyanate phalloidin and fluorimetric measurements were performed in single couplets using a scanning laser cytometer, ACAS 570. F-actin distribution was assessed by the ratio of canalicular area fluorescence/total couplet fluorescence (CF/TF). The organization of the F-actin filaments was restored in IRHC 3-6 h after plating. At non-cytotoxic concentrations, most cholestatic compounds induced a significant accumulation of F-actin around the bile canaliculus as indicated by increased fluorescence in the pericanalicular area and by the increased CF/TF ratio as compared to the controls. This accumulation could be a consequence of an inhibition of F-actin depolymerization or a higher organization of actin (redistribution, bundling or reorientation). Hepatotoxic but non-cholestatic compounds did not induce any change in pericanalicular F-actin. Abnormalities of pericanalicular F-actin therefore appear to be a specific marker of hepatocellular cholestasis.