Heat stress (HS) causes reductions in milk yield and content of fat and protein. It is not clear whether these losses are due to reduced number or activity (or both) of mammary epithelial cells. To test mechanisms by which mammary metabolism is impaired by HS, MAC-T cells were incubated in different ambient temperature conditions: thermo-neutral (TN; 37°C) and heat stress (HS; 42°C). In both conditions, 3 mediums varying in amino acid (AA) concentrations were used. These media were: optimal amino acid profile as control (Con), control plus methionine (Met), and control plus arginine (Arg). Consequently, there were 6 treatment combinations: TN-Con, TN-Met, TN-Arg, HS-Con, HS-Met and HS-Arg. After incubation, cells were harvested and RNA was extracted for the study of gene expression by quantitative RT-PCR. Both HS and AA increased (P < 0.01) the expression of heat shock proteins (HSP70A1A), transcription and translation factors (RPS6KB1, JAK2, AKT2), and AA transporters (SLC1A5, SLC7A1). The expression of PPARG (fat transcription regulation), FASN (de novo fatty acids synthesis), BCL2L1 (anti-apoptotic), and AKT1 (cell survival) decreased (P < 0.05) with HS, but increased (P < 0.05) with AA. Furthermore, HS downregulated ACACA (de novo fatty acids synthesis) and upregulated BAX (apoptotic). Supplementation with Met or Arg upregulated (P < 0.05) genes related to transcription and translation (MAPK1, MTOR, SREBF1), cell proliferation (MKI67), and insulin signaling (IRS1). The expression of EIF4EBP1 (inhibitor of protein synthesis) was upregulated by HS, but downregulated by Met. Results suggest that heat stress exerts its negative effect on milk production at least in part by inhibiting mammary synthetic capacity as well as increasing apoptosis of mammary cells without affecting cell proliferation. Supplementation with AA (especially Met) increased mammary synthetic activity and had a positive effect on cell number by increasing proliferation and decreasing apoptosis. This raises the possibility that supplemental rumen-protected Met during heat stress might have a positive effect on mammary metabolism.