Twenty-seven strains of Listeria monocytogenes previously isolated from food (n=16) and human patients of listeriosis (n=11) were characterized and compared based on their ability to survive through the simulated gastrointestinal tract conditions. Cells were exposed (60 or 120min) to low pH in the presence of pepsin, to simulate the digestion in the stomach, and subsequently to bile salts to simulate the digestion in the small intestine (60 or 120min). Their survival was shown to be origin- (food and clinical) and strain dependent (p<0.001) and also significantly dependent on the imposed simulated gastric conditions (long vs. quick exposure) (p<0.001). In comparison to the food isolates, the clinical strains were in general more resistant and survived better to the two challenges imposed. Some of the tested strains, after the exposure to low pH in the presence of pepsin, became injured and subsequently more susceptible to the bile salts challenge. It was demonstrated that one of the most important natural barriers against foodborne pathogens might not be effective since it was shown that L. monocytogenes isolates that survived through the pH challenge were also able to survive the subsequent challenge to bile salts.