Experiments are presented to determine the effect of labeled precursor amino acid on synthesis of beta-lactoglobulin and beta-casein and to identify the most limiting amino acids in Eagle's minimal essential medium for synthesis of milk protein. Protein synthesis was measured by incorporation of carbon-14 labeled amino acids into milk protein from bovine mammary cell culture. Mammary tissue from 13 Holsteins (12.7 kg milk/day) was dispersed and cultured for 18 h. In Experiment 1, three replications (cows) with three amino acid mixtures, utilizing labeled lysine or leucine as the marker amino acid, showed that labeled lysine overestimated the effect of medium lysine, and labeled leucine overstimated the effect of leucine on beta-lactoglobulin and beta-casein synthesis. Subsequently Experiment 2 utilized 10 replications (cows) to determine the effect of a three-fold increase of each of 13 amino acids in Eagle's medium. Increased threonine or methionine improved beta-lactoglobulin synthesis and cystine increased beta-casein. Second and third best response in beta-casein were from addition of methionine and threonine. Combined analysis from 10 cows and two protein responses by multivariate procedures identified, in order of decreasing magnitude, cystine, threonine, and methionine as limiting amino acids in the dispersed cell culture.