The goal of the current study was to clarify whether commercially available probiotics induce greater trans-epithelial activation of human leukocytes than do commensal, food-derived and pathogenic bacteria and to identify the compounds responsible for this activation. Eleven different bacterial strains, and some of their pathogen-associated molecular patterns, were incubated apically on a confluent layer of intestinale pithelial cells(Caco-2), which were basolaterally co-cultured with human mononuclear leukocytes. Only Gram-negative bacteria having Enterobacteriaceae-type endotoxin (commensal Escherichia coli K12, probiotic E. coli Nissle, EPEC) induced basolateral production of TNF-α, IFN-γ, IL6, 8, and10. Gram-positive probiotics (Lactobacillus spp. and Bifidobacterium spp.) had virtually no effect. In addition, commensals (Enterococcus faecalis, Bacteroides vulgatus) and food fermenters (Lactobacillus spp.) did not stimulate leukocytes transepithelially. Endotoxin from E. coli and Salmonella enterica Ty-phimurium induced comparable transepithelial stimulation of leukocytes, but not endotoxin from B. vulgatus or lipoteichoic acid from E. faecalis. Endotoxin-binding agents (polymyxin, colistin) completely abrogated transepithelial activation of  leukocytes. Enterobacteriaceae-type endotoxin is a crucial factorin transepithelial stimulation of leukocytes, regardless of whether it is produced by probiotics or other bacteria.  Hence, transepithelial stimulation of leukocytes’ innate immune response seems to not be linked to the health-promoting effects of probiotics.