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The amino acid requirements of Neocallimastix sp N 13 isolated from sheep rumen were studied using supplements containing either ten essential amino acids (10 EAA) or eight nonessential amino acids (8 NEAA), or both (18 AA). Although the fungus could grow in a medium containing ammonium sulphate as the sole source of nitrogen, amino acid supplements, especially 18 AA, greatly stimulated its growth. Omission of the sulphur-containing amino acids (SCAA) from the 18 AA mixture markedly reduced the growth-stimulating effect, and sulphide, but not sulphate, substituted for SCAA only partly compensated for this omission. Omission of aromatic, branched chain, basic, acidic, aliphatic and hydroxy amino acids, and proline, singly or in combination, all reduced fungal growth to some degree as compared with the 18 AA supplement. A three amino acid (leucine, methionine and histidine) supplement was a potent stimulator for the fungus, whereas another three amino acid combination (glutamic acid, methionine and serine) was ineffective. The results indicated that Neocallimastix sp N 13 effectively utilized amino acids for its growth, but the requirements for the different amino acids differed from those of N patriciarum.