Aim: To assess the role of lactate as a precursor for butyrate biosynthesis in human colonic microflora. Methods and Results: Three human faecal microfloras were incubated in vitro with media supplemented with 30 mmol 1(-1) unenriched or C-13-enriched lactate. Lactate metabolism and short-chain fatty acid (SCFA) production were quantified. Lactate conversion to butyrate was investigated by gas chromatography-mass spectrometry and the pathways involved were identified by C-13 nuclear magnetic resonance spectroscopy. All human faecal microfloras rapidly and completely fermented lactate, yielding approx. 19 mmol 1(-1) total SCFAs. However, the SCFA composition varied markedly between microfloras. Butyrate was the main end-product for two microfloras but not for the third (60 and 61% vs 27% of the net concentration of SCFA produced respectively). The latter was typified by its ability to produce propionate as a major product (37%), and valerate (3%). C-13-Labelling showed that butyrate was produced through the acetyl-CoA pathway and that the three microfloras possessed significant differences in their metabolic pathways for lactate consumption. Conclusions: In contrast to the ruminal microflora, the human intestinal microflora can utilize both D- and L-lactate as precursors for butyrate synthesis. Inter-individual variation is found. Significance and Impact of the Study: This study suggests that the butyrogenic capability of colonic prebiotics could be related to lactate availability. These findings will direct the development of selection strategies for the isolation of new butyrate-producing bacteria among the lactate-utilizing bacteria present in the human intestinal microfloras.