Extracellular amino acid (AA) profile may affect intracellular AA concentrations and profile as well as signaling proteins that regulate translation rate. The objective of this work was to assess the effects of various extracellular AA profiles and Lys to Met ratio to determine signaling protein sensitivity. Six AA profiles of DMEM, blood meal (BM), corn gluten meal (CM), casein (CS), blood plasma of cows milking 45 kg/d (CW), and a negative control (NC) represented the profile factor (AAPROF) and Lys/Met ratio unchanged or set to 3:1 was the Lys/Met factor (ML) for a total of 12 treatments with 4 replications. The concentrations of total AA for all treatments except NC (0 mg/L) were set to 659 mg/L (63% of DMEM) which previously was shown to result in maximal stimulation of casein synthesis. Confluent mammary epithelial cells were exposed to treatments for 75 min. Intracellular concentrations of Met, Lys, Leu, Ile, and Thr were affected by AAPROF (P < 0.02) whereas only Met and Lys were affected by ML, increasing by 13.6 μmol/L and 11.5 μmol/L (P < 0.01). Intracellular Met and Lys concentrations were 145 and 274% (P < 0.01) greater for the NC and ML at 3:1 ratio treatment versus other ML 3:1 ratio treatments despite similar extracellular concentrations indicating greater uptake. Within mTOR pathway, mTOR, ribosomal protein S6 kinase 1 (S6K1), and eukaryotic initiation factor 4 E binding protein 1 were induced by AAPROF (P < 0.01) while only S6K1 was affected by ML (P = 0.11). mTOR pathway proteins had greater phosphorylation for DMEM, BM, CM, CS, and CW versus NC (P < 0.01). Blood meal (P = 0.15) and CM (P = 0.06) had higher phosphorylation than CS and CM tended to be higher than CW (P = 0.14) for S6K1 respectively. For mTOR, BM, CS, and CM tended to have higher phosphorylation than CW (P = 0.07). Eukaryotic initiation factor 2 α subunit was unaffected by PROF and ML factors (P = 0.23). Setting Lys/Met at a 3:1 ratio had a small positive effect on S6K1 regardless of AA profile. Changes in extracellular AA profiles largely translated to intracellular AA and these varying profiles in general stimulated mTOR pathway related proteins.