Earlier, we have shown that spontaneously isolated non-pathogenic bacteria Serratia grimesii and Serratia proteamaculans invade eukaryotic cells, provided that they synthesize thermolysin-like metalloproteases ECP32/grimelysin or protealysin characterized by high specificity towards actin. To address the question of whether the proteases are active players in entry of these bacteria into host cells, in this work, human larynx carcinoma Hep-2 cells were infected with recombinant Escherichia coli expressing grimelysin or protealysin. Using confocal and electron microscopy, we have found that the recombinant bacteria, whose extracts limitedly cleaved actin, were internalized within the eukaryotic cells residing both in vacuoles and free in cytoplasm. The E. coli-carrying plasmids without inserts of grimelysin or protealysin gene did not enter Hep-2 cells. Moreover, internalization of non-invasive E. coli was not observed in the presence of protealysin introduced into the culture medium. These results are consistent with the direct participation of ECP32/grimelysin and protealysin in entry of bacteria into the host cells. We assume that ECP32/grimelysin and protealysin mediate invasion being injected into the eukaryotic cell and that the high specificity of the enzyme towards actin may be a factor contributed to the bacteria internalization.