Requirement of nonessential amino acids by animal was investigated with two mouse myeloma cell lines (MPC-11 and NS-1) and a human globular glanule cell line (K562) cultured in vitamin B6-deleted serum-free medium. All cell lines did not show any requirement for such 7 amino acids as alanine, asparagine, aspartic acid, glutamic acid, glycine, proline and serine in the presence of vitamin B₆. When vitamin B₆ was deleted from the medium, MPC-11 cells required strongly alanine, serine and glycine, in that order, whereas NS-1 and K562 cells required only alanine and serine. These results suggested that the effect of vitamin B₆ on amino acids metabolism differed according to the kind of cells and verified the usefulness of serum-free cell culture method for the investigation of cellular nutrition.