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, six healthy exclusively breast-feeding women [27 +/- 3 yr; body mass index (BMI) 26 +/- 2 kg/m2] between 6 wk and 3 mo postpartum and six healthy nonlactating women (28 +/- 2 yr; BMI 22 +/- 1 kg/m2) were studied two times, 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 AA 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.