We investigated the effect of some eucaryotic cytoplasmic structure and function inhibitors on the entry into HeLa cells of the Escherichia coli HB101 K12 strain, harbouring the recombinant plasmid pRI203, in which is cloned a 3.2 Kb chromosomal fragment of Yersinia pseudotuberculosis. Substances impairing microfilament structures and functions (cytochalasin B and trifluoroperazine) significantly reduced invasion ability whereas microtubule organization inhibitors (colchicine and vinblastine) were ineffective. Data obtained with a lipophilic weak base (methylamine), which raises the pH of intracellular vesicles, demonstrated that, in entry pathway of E. coli HB101 (pRI203), endosome acidification is not required. Host cell energy has been shown to contribute to bacterial internalization since the presence of oxidative phosphorylation and glycolysis inhibitors (sodium azide, 2-dinitrophenol and 2-deoxy-D-glucose) during the invasion process, affected bacterial entry.