The concentrations of adenosine 5′-mono-, -di-, and -triphosphate (AMP, ADP and ATP) in sheep digesta were measured by a new bioluminescence method. ATP in rumen digesta varied with time after feeding and diet, and most markedly according to the size of the population of ciliate protozoa: the ATP content of rumen digesta from ciliate-free sheep was less than a quarter of that of faunated animals receiving the same diet. The adenylate energy charge (EC) (ATP + ADP/[ATP + ADP + AMP]), an indicator of metabolic activity, was high (0·77–0·94) in all rumen samples, but did not appear to be as useful a measurement of activity as ATP alone. As digesta passed along the alimentary tract, the ATP content decreased progressively, from 0·9–2·2 μmol/g dry matter in the rumen to 0·5–1·0μmol/g in the abomasum and duodenum, 07middot;03–0·08 μmol/g in ileal digesta and 0·01–0·02 μmol/g in faeces. EC also tended to decrease, further emphasizing the large decrease in microbial activity which occurs in the hindgut.