Amino acids stimulate the growth rate and growth yield of ruminal micro-organisms, but the basis of this stimulation, in terms of amino acids which most limit growth, has never been fully established. Here, for the first time, a deletion approach was investigated using in vitro incubations of mixed ruminal micro-organisms supplied with a mixture of xylose, starch and cellobiose as energy sources and ammonia plus a complete amino acids mixture or mixtures with a single amino acid omitted as nitrogen sources, enabling the evaluation of the impact on ruminal fermentation of the deletion of a single amino acid from a complete amino acids mixture. Significant effects (P<0.05) on total gas production were observed after 10 h of incubation when glutamate, glutamine, isoleucine, leucine, phenylalanine, serine, tryptophan or tyrosine were deleted from the amino acids mixture. The only significant effect of an amino acid deletion on volatile fatty acid production at 10 h was with serine (P<0.05), although the effect of omitting others, including arginine, isoleucine, leucine and phenylalanine, approached significance (P<0.01). The removal of leucine caused a 0.09 decrease in growth yield (P<0.05); no other deletion affected the yield significantly (P>0.05). Net gas production for each treatment was calculated by subtracting gas production in the absence of carbohydrates from gas production in their presence, thus eliminating gas production from amino acids from the values. At all times up to 10 h, the most significant effects on net gas production were found when serine, leucine, or the aromatic amino acids were omitted from the amino acids mixture. Thus, the deletion approach confirmed that no single amino acid limits ruminal fermentation more than any other, although a few, principally phenylalanine, leucine and serine, have a particularly significant role in the ruminal fermentation rate of soluble, rapidly degraded materials and/or microbial growth efficiency.