1. A method is described for the estimation of the microbial and dietary protein entering the small intestine of sheep, based on an intraruminal infusion of [35S]sulphate, and isolation of the labelled microbial protein.
2. Samples of the microbial fraction contained in duodenal digesta were isolated by highspeed centrifugation and a scheme of analysis was devised for the determination of the specific activity of the methionine in the microbial fraction and the duodenal digesta.
3. In the absence of dietary methionine in duodenal digesta, it was postulated that the ratio of the specific activities of microbial methionine (M) and digesta methionine (D) should equal 1. Using two purified diets containing urea as the sole source of nitrogen, the M:D ratio was found to vary from 0·94 to 1·08 (mean 1·01 ± 0·01).
4. Addition of known quantities of dietary protein to labelled duodenal digesta obtained from one sheep fed on a non-protein diet increased the M:D ratio. Prediction of the dietary protein present from changes in the M:D ratio gave satisfactory agreement with the amounts of dietary protein known to be present (96–101% of known value, mean 98·4 ± 1·24%).
5. When animals were fed hourly the M:D ratio remained reasonably constant throughout 24 h. In contrast, with twice-daily feeding the M:D ratio varied markedly throughout the 24 h. However, summation of the estimates made of the flow of microbial and dietary protein into the duodenum in each 2 h period throughout 24 h agreed closely with the estimates made from samples withdrawn from the accumulated flow of digesta over the whole 24 h period.
6. The method was finally adapted to situations with either frequent or infrequent feeding patterns, and the need for a priming infusion period of 16 h before the collection and sampling of duodenal digesta was established.