Several methods have been developed for the determination of the proportion of nitrogen-containing substances of microbial origin in the digestive tracts of cattle. These include assays that use nucleic acids and adenosine triphosphate as indicators, radioisotopes ³⁵S, ¹⁵N, ³²P and ³³P incorporated into bacterial protein and phospholipids, duodenal amino acid composition with amino-ethylphosphonic acid, diaminopimelic acid and D-alanine (D-Ala) contents as indicators. On the basis of the data in the literature on D-amino acid content of milk and milk products the authors came to the conclusion that D-glutamic acid (D-Glu) and D-aspartic acid (D-Asp) can be considered as markers for proteins of bacterial origin. To demonstrate this, experiments have been carried out and some of the results are reported here. © 2001 Elsevier Science B.V.