A deletion approach was undertaken to identify which amino acids (AA) most limited the growth of mixed ruminal microorganisms on xylan. Ruminal fluid was withdrawn from sheep receiving a mixed grass hay/concentrate diet and incubated for 24 h with oat spelts xylan in the presence or absence of a mixture of 20 AA or the same mixture with a single AA deleted. Gas and volatile fatty acid production were increased by the AA mixture in comparison with incubations in which ammonia was the only added nitrogen (N) source, and the deletion of each of the aromatic AA, tyrosine, phenytalanine and tryptophan, as well as leucine and methionine, led to decreases (P < 0.05) in fermentation rate. The addition of aromatic AA as a mixture to ammonia-only fermentations increased (P < 0.05) the fermentation rate but failed to replicate the benefits of the complete mixture of AA. Although the addition of all 20 AA increased (P < 0.05) the microbial yield by up to 0.56, no single AA deletion had a significant (P > 0.05) influence on microbial yield, and the aromatic AA mixture also did not increase the microbial yield on xylan over the yield with ammonia as sole N source. It was concluded that aromatic AA may be first-limiting for xylan fermentation, but they cannot replace the benefits of a complete mixture of 20 AA in stimulating xylan fermentation by ruminal micro-organisms.