The gastrointestinal tract of neonates becomes colonized immediately after birth with environmental microorganisms, mainly from the mother; strong evidence suggests that the early composition of the microbiota of neonates plays an important role for the postnatal development of the immune system. The present study was designed to evaluate by means of a molecular biology approach the relation between the intestinal ecosystem of the newborn and the mode of delivery. The intestinal bacterial composition on d 3 of life was investigated in 23 infants born by vaginal delivery and in 23 infants delivered by cesarean section. PCR-denaturing gradient gel electrophoresis and PCR-temperature gradient gel electrophoresis have been utilized, together with the specific amplifications for 10 Bifidobacterium species, 3 Ruminococcus species, and Bacteroides. The intestinal microbiota of neonates delivered by cesarean delivery appears to be less diverse, in terms of bacteria species, than the microbiota of vaginally delivered infants. The intestinal microbiota after cesarean delivery is characterized by an absence of Bifidobacteria species. Vaginally delivered neonates, even if they showed individual microbial profiles, were characterized by predominant groups such as B. longum and B. catenulatum. Our data demonstrate that the mode of delivery has a deep impact on the composition of the intestinal microbiota at the very beginning of human life. This study opens the path to further investigations to confirm the link between microbiota composition and immune system development and to identify tools for the modulation of the intestinal microbiota of cesarean-delivered neonates. Additionally, we underline the importance of adequate microbiological tools used to support clinically relevant trials, if intestinal microbiota is considered as a study outcome.