Flavomycin is an antibiotic that promotes growth in ruminant and non-ruminant livestock. The aim of this study was to determine the mechanism of action of flavomycin in sheep by measuring microbial numbers, microbial metabolism and gut tissue protein turnover at different sites in the digestive tract. Two weight-matched groups (n 5) of male castrate lambs (30 kg) received 800 g grass cubes/d for 6 weeks, with one group receiving 20 mg/d flavomycin during the last 2 weeks. Samples of digesta and gut tissue segments were obtained immediately post mortem, 90 min after a flood-dose of [ring-D5]phenylalanine. Viable bacterial counts and volatile fatty acid concentrations were highest in ruminal digesta, followed by the colon and caecum, then the duodenum and ileum. The only effect of flavomycin was an increased bacterial count in the rumen (3.5 v. 1.2 x 10(9) per g; P=0.04). Acetate was proportionally greater and propionate and butyrate were lower in the caecum and colon than the rumen. Flavomycin had no effect on volatile fatty acid proportions or ammonia concentrations. Bacteria growing on peptides as sole C source were not affected by flavomycin. Proteolytic, peptidolytic and amino acid deamination activities were similar in the rumen, caecum and colon; they tended to be lower in animals receiving flavomycin. Protein turnover in ruminal wall and duodenal tissues, measured by a flood-dose technique, decreased with flavomycin (P=0.075 and 0.027, respectively). Thus, flavomycin differs from ionophores in its mode of action. It may influence protein metabolism of both digesta and tissue throughout the ruminant digestive tract.