The pH of the colonic lumen varies with anatomical site and microbial fermentation of dietary residue. We have investigated the impact of mildly acidic pH, which occurs in the proximal colon, on the growth of different species of human colonic bacteria in pure culture and in the complete microbial community. Growth was determined for 33 representative human colonic bacteria at three initial pH values (approximately 5.5, 6.2 and 6.7) in anaerobic YCFA medium, which includes a mixture of short-chain fatty acids (SCFA) with 0.2% glucose as energy source. Representatives of all eight Bacteroides species tested grew poorly at pH 5.5, as did Escherichia coli, whereas 19 of the 23 gram-positive anaerobes tested gave growth rates at pH 5.5 that were at least 50% of those at pH 6.7. Growth inhibition of B. thetaiotaomicron at pH 5.5 was increased by the presence of the SCFA mix (33 mM acetate, 9 mM propionate and 1 mM each of iso-valerate, valerate and iso-butyrate). Analysis of amplified 16S rRNA sequences demonstrated a major pH-driven shift within a human faecal bacterial community in a continuous flow fermentor. Bacteroides spp. accounted for 27% of 16S rRNA sequences detected at pH 5.5, but 86% of sequences at pH 6.7. Conversely, butyrate-producing gram-positive bacteria related to Eubacterium rectale represented 50% of all 16S rRNA sequences at pH 5.5, but were not detected at pH 6.7. Inhibition of the growth of a major group of gram-negative bacteria at mildly acidic pH apparently creates niches that can be exploited by more low pH-tolerant microorganisms.