Background:Characterising the regulation of milk component synthesis in response to macronutrient supply is critical for understanding the implications of nutritional interventions on milk production. Gene expression in mammary gland secretory cells was measured using RNA isolated from milk fat globules from 6 Holstein-Friesian cows receiving 5-d abomasal infusions of saline, essential amino acids (AA), or glucose (GG) or palm olein (LG) without (LAA) or with (HAA) essential AA, according to a 6 × 6 Latin square design. RNA was isolated from milk fat samples collected on d 5 of infusion and subjected to real-time quantitative PCR. We hypothesised that mRNA expression of genes involved in de novo milk fatty acid (FA) synthesis would be differently affected by GG and LG, and that expression of genes regulating transfer of tricarboxylic acid cycle intermediates would increase at the HAA level. We also hypothesised that the HAA level would affect genes regulating endoplasmic reticulum (ER) homeostasis but would not affect genes related to the mechanistic target of rapamycin complex 1 (mTORC1) or the integrated stress response (ISR) network. Results:Infusion of GG did not affect de novo milk FA yield but decreased expression of FA synthase (FASN). Infusion of LG decreased de novo FA yield and tended to decrease expression of acetyl-CoA carboxylase 1 (ACC1). The HAA level increased both de novo FA yield and expression of ACC1, and tended to decrease expression of mitochondrial phosphoenolpyruvate carboxykinase (PCK2). mRNA expression of mTORC1 signaling participants was not affected by GG, LG, or AA level. Expression of theεsubunit of the ISR constituent eukaryotic translation initiation factor 2B (EIF2B5) tended to increase at the HAA level, but only in the presence of LG. X-box binding protein 1 (XBP1) mRNA was activated in response to LG and the HAA level. (Continued on next page)(Continued from previous page) Conclusions:Results show that expression of genes involved in de novo FA synthesis responded to glucogenic, lipogenic, and aminogenic substrates, whereas genes regulating intermediate flux through the tricarboxylic acid cycle were not majorly affected. Results also suggest that after 5 d of AA supplementation, milk protein synthesis is supported by enhanced ER biogenesis instead of signaling through the mTORC1 or ISR networks.