The objective of this study was to evaluate the effects of jugular infusion of 2 groups of AA on essential AA availability and utilization by the mammary glands. Four cows (78 ± 10 DIM) were assigned to 4 jugular infusion treatments: saline (CON), methionine plus lysine plus histidine (MKH), isoleucine plus leucine (IL), or MKH plus IL (MKH-IL) in a 4 × 4 Latin square design. Each period was 16 d in length with 8 d of adaption followed by 8 d of jugular AA infusion. Infusion rates were 10 g of methionine, 38 g of lysine, 20 g of histidine, 50 g of leucine and 22 g of isoleucine per day. On last day of each period, a ¹³C labeled AA mix was infused into the jugular vein over a 6 h period, and blood samples were collected hourly and assessed for AA enrichment. Cows were fed a basal diet consisting of 15.2% crude protein with adequate rumen degradable protein but 15% deficient in metabolizable protein. Milk production increased significantly with infused IL (P = 0.04), but did not change in response to MKH (P = 0.44). Milk protein concentration and yield increased for the MKH infusion (P < 0.01) whereas milk protein yield tended to increase for IL (P = 0.08). Total plasma AA entry rates were estimated for each EAA by fitting a 6-pool, dynamic model to observed plasma, ¹³C AA enrichment. The blood Met entry rates for CON, MKH, IL and MKH-IL were 57, 82, 62, 90 g/d respectively after subtracting infused Met. MKH infusion significantly increased Met entry (P < 0.01) and IL infusion tended to increase Met entry (P = 0.06), which was related to greater Met intake (P = 0.09). The increase in Met entry with MKH infusion reflected increased Met availability from the diet or increased recycling of Met in the body. Essential AA transport and metabolism by the mammary glands will be reported.