The partitioning of dietary and endogenous nutrients during lactation is not well understood. To examine associations between plasma hormone and substrate profiles and indices of either maternal body protein metabolism or lactational performance, we measured plasma insulin, cortisol, prolactin, thyroxine, triiodothyronine, individual amino acid, blood urea nitrogen, and prealbumin concentrations in lactating and nulliparous women in the postabsorptive state. We related these measurements to the subjects' nitrogen balance, urinary 3-methylhistidine excretion, [1-13C]leucine metabolism and milk production. Insulin concentrations showed significant positive relationships with nitrogen balance and prealbumin concentrations; cortisol levels showed a significant negative relationship with nitrogen balance and a significant positive relationship with leucine incorporation into protein. Thyroid hormone concentrations showed significant positive relationships with urinary 3-methylhistidine excretion, leucine incorporation into protein, and milk production. Proline concentrations were associated positively with nitrogen balance and negatively with leucine incorporation into protein, whereas glutamate-glutamine concentrations showed positive associations with leucine oxidation and milk nitrogen concentrations. We propose that insulin and cortisol modulate the channeling of nutrients between anabolic and anti-anabolic aspects of maternal body protein metabolism, whereas thyroid hormones and cortisol modulate nutrient partitioning toward milk production and visceral protein synthesis. We suggest that some nonessential amino acids (proline, glutamate-glutamine) may become limiting during lactation because of their unique contributions to milk protein synthesis.