Technical Abstract: Little is known about amino acid (aa) and protein metabolism in lactating women. We hypothesized: 1) aa sources other than the plasma acid pool provide substrate for milk protein synthesis in humans; and 2) if albumin was one such source, then albumin fractional synthesis rate (FSR) is higher in the lactating women. To test these hypotheses, 6 healthy exclusively breastfeeding women (27+/-3 y; BMI 26+/-2 kg/m2) between 6 wks and 3 months postpartum, and 6 healthy non-lactating women (28+/-2 y; BMI 22+/-1 kg/m2) were studied twice, in random order, during 22 h fasting or 10 h of continuous feeding with a mixed nutrient drink. Protein metabolism was determined using [1-13C]leucine and [15N2]urea. In both the fed and fasted states, a significant portion of milk protein (20+/-5 and 31+/-6%, respectively) was derived from sources other than the plasma free aa pool. A 70% higher (p<0.02) FSR of albumin was observed in lactating women during feeding, suggesting that albumin is a likely source of aa for milk protein synthesis. We conclude that plasma free amino acids contribute only 70-80% of the substrate for milk protein synthesis in humans and that albumin may be a significant source of amino acids for the remainder.