The amino acid requirement for milk protein synthesis was investigated in the rat mammary cells in tissue culture. Studies involved: 1) the amino acid reduction from a positive control (3X) back to a negative control (1X) for each of the essential amino acids that were present in the 1X-MEM (Omission Study) [X refers to the amino acid concentration of Eagle's minimal essential medium (MEM)], 2) the addition experiments which resulted in an inverse relation to those of the Omission studies, and 3) a three-dimensional central composite experiment with three variables (X₁ = lysine; X₂ = methionine, valine and arginine; X₃ = isoleucine, tryptophan, threonine, phenylalanine and histidine), embracing 15 treatment combinations. In addition, supplemental studies were conducted which determined: 1) the effect of varied labeled amino acid precursors (¹⁴C-U-lysine, phenylalanine and leucine) on the measured synthesis of milk protein, 2) the effect of varying cell density (6 x 10⁶ to 6 x 10⁷) on protein synthesis, and 3) the response to graded amino acid complement (1X through 5X) on milk protein synthesis. Supplemental studies indicated that: 1) synthesis of β-lactoglobulin and β-casein in response to the labeled amino acids were significant (P<.05), 2) cell numbers between 6 x 10⁶ to 6 x 10⁷ did not significantly (P>.05) alter the rates of the milk protein synthesis, and 3) the response of amino acid complement addition to the 1X-MEM was linear (P<.01) for β-lactoglobulin and β-casein synthesis. Data analysis from Omission and Addition experiments revealed that: 1) the first limiting amino acid was lysine, 2) the second limiting group of amino acids were methionine, valine and arginine, and 3) the least limiting amino acids were tyrosine, leucine and glutamine. The response surface study determined that the optimal combination of essential amino acids for the maximum milk protein synthesis were: 1) lysine = 15X, 2) methionine, valine and arginine = 4.5X, and 3) isoleucine, tryptophan, threonine, phenylalanine and histidine = 1. 5X elevation over that present in the 1X-MEM, and 4) leucine, tyrosine, cystine and glutamine = 1X. On the basis of these findings, it was possible to construct the optimal essential amino acid complement unique to the rat mammary cells in tissue culture for the maximum milk protein synthesis. / Ph. D.