Summary: Analysis of human gut contents showed that substantial quantities of soluble protein, ammonia and branched chain volatile fatty acids occurred throughout the large intestine [0·1-24·4 g (kg contents)−1, 7·7-66·0 mmol (kg contents)−1 and 1·5-11·1 mmol (kg contents)−1 respectively]. The presence of these metabolites suggested that substantial proteolysis was occurring. In vitro studies showed that casein and bovine serum albumin were partly degraded in slurries of human faeces over a 96 h incubation period, to produce TCA-soluble peptides, ammonia and volatile fatty acids. Proteolytic activity detected in the stools of five individuals ranged from 3·5 to 19·8 mg azocasein hydrolysed h−1 (g faecal material)−1. Washed cell and washed particulate faecal fractions accounted for 24-67% of total activity. The predominant proteolytic bacteria in the faecal samples examined were identified as Bacteroides spp. [1·0 × 1011-1·3 × 1012 (g dry wt faeces)−1] and Propionibacterium spp. [1·2 × 108-1·0 × 1010 (g dry wt faeces)−1]. Other proteolytic bacteria which occurred in lesser numbers were identified as belonging to the genera Streptococcus, Clostridium, Bacillus and Staphylococcus. These results demonstrate that the gut microflora could potentially play a major role in proteolysis in the human colon.