The objective of this study was to test whether mammary AA metabolism and blood flow (BF) fluctuate in relation to changes in milk protein yield brought about by unilateral milking frequency. To investigate these relationships, the vascular systems of the mammary glands of 4 lactating goats were separated by ligation and flow probes were fitted around the external pudic artery of each gland. Goats were subjected to 3 consecutive periods (5 d each) during which 1) both glands were milked twice daily (2x; period 1); 2) glands were unilaterally milked, with gland A milked 8 times daily and gland B milked incompletely (30% of period 1 milk yield retained in the gland) once daily (8x vs. 1x; period 2); and 3) both glands milked twice daily (2x; period 3). On d 5 of periods 1 (2x) and 2 (8x vs. 1x), arteriovenous [1-(13)C]Leu kinetics were monitored for each gland. Milk protein yield was greater for gland A compared with gland B. After 2x milking resumed in period 3, yields for both glands returned to period 1 levels. Similarly, BF increased (19%) for gland A (8x), whereas BF was decreased (21%) for gland B (1x). For both glands, BF returned to previous levels when 2x milking resumed in period 3. Mammary net uptakes and oxidation of Leu were greater for the gland milked 8x compared with that milked 1x. Net uptakes from blood of Ile, Leu, Val, Lys, Pro, and Arg were greater (39-79%) for the 8x compared with the 1x gland. In conclusion, the excess removal and oxidation by the mammary gland of some amino acids appears an obligatory requirement to support increases or decreases in milk protein yield when milking frequency is altered.