Due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (BT) across the intestinal epithelial barrier are poorly understood. Thus, we have utilized the Caco-2 intestinal cell line to study this process. Caco-2 cells were grown to confluence on semipermeable membrances contained in the upper compartment of a 2 compartment system. Cellular confluence and tight junction integrity was verified by measurements of the transepithelial electrical resistance in ohms cm2, BT was measured by culturing the bacteria (nonpathogenic Escherichia coli) that were able to cross the Caco-2 monolayer and were present in the bottom compartment, as well as by monitoring the passage of 1-μm fluorescent beads. Caco-2 cells were pretreated with several metabolic inhibitors: 1.0 mM sodium azide (oxidative phosphorylation), 10 μg/ml nocodazole (microtubule), 10 μM phalloidine (microfilament), and 5.0 μg/ml cytochalasin D (microfilament). To investigate the mechanisms of BT. Both bacteria and fluorescent beads crossed the Caco-2 monolayer. Azide had no effect on BT, while both nocodazole (n = 17) and phalloidine (n = 14) significantly decreased translocation of E. coli versus control monolayers (p < .05). Cytochalasin D increased BT versus control membrances, however this was associated with loss of tight junction integrity (transepithelial electrical resistance decreased from 201 ± 79 to 87 ± 6.4). BT across Caco-2 cells appears to be a polar process which is to some extent microtubule- and microfilament-dependent.