In the past years several methods have been developed for the determination of the proportion of the nitrogen-containing substances of microbial origin passed from the rumen into the abomasum or the small intestine. Recently, on examining the D-amino acid content of foodstuffs, particularly milk and milk products, it has been observed that, in addition to D-Ala, D- glutamic acid (D-Glu) and D-aspartic acid (D-Asp) can also be detected in similar quantities, primarily in products which have links with bacterial activity. This gave rise to the idea of examining the diaminopimelic acid (DAPA), D-Glu and D-Asp content of bacteria extracted from the rumen of cattle and that of chyme from the same cattle, in order to determine the type of relation existing among these three components, and to establish whether D-Asp and D-Glu can be used in the estimation of protein of bacterial origin. On determination of the DAPA, D-Asp and D-Glu content by means of amino acid analyser and high performance liquid chromatography of duodenal chyme from five growing bulls and of ruminal bacteria from the same bulls, the following values were established. For chyme (and, in brackets, for ruminal bacteria) r value calculated by means of linear regression was 0.78 (0.76) between DAPA and D-Asp, and 0.70 (0.81) between DAPA and D-Glu. The r values between the crude protein content of ruminal bacteria and the markers examined were found to be the following: DAPA, 0.74; D-Asp, 0.73; D- Glu, 0.61. In the model experiment performed for the re-obtaining of values for protein of bacterial origin the theoretical values were determined on the basis of D-Asp and D-Glu and values approximately 10% higher than the theoretical value on the basis of DAPA. It is therefore recommended that in addition to DAPA these other two amino acids be included among the bacterial protein markers.