Erythropoietin (Epo) is a normal constituent of human milk, but the origin and fate of this cytokine in milk are not known. Regarding its origin, we hypothesized that cells of the mammary gland secrete Epo into milk actively and, therefore, that concentrations in milk do not correlate with concentrations in serum. Regarding its fate, we hypothesized that Epo concentrations in milk change with time postpartum and that Epo in milk is protected from digestion in the neonatal gastrointestinal tract. To address these issues, we measured Epo concentrations in 103 milk samples (ELISA), 55 of which were paired with serum. Mammary duct epithelial cells were evaluated as a source of Epo by breast tissue immunohistochemistry and by cell culture. Circulating and milk Epo were compared by Western analysis to detect size differences, possibly reflecting differences in processing. Epo stability in simulated conditions of digestion was evaluated. We observed that milk Epo concentrations increase as a function of duration of breast-feeding and have a negative correlation with serum Epo or milk protein concentration. Mammary duct epithelial cells from breast biopsies of lactating women had marked immunoreactivity to Epo, but such activity was minimal to absent in nonlactating breast tissue. Further evidence that mammary duct epithelia produce Epo was obtained by observing Epo mRNA and protein expression in cultured human mammary epithelial cells. The molecular size of Epo in milk and serum is identical. Recombinant Epo added to human milk or commercial infant formulas was relatively stable in conditions that simulate gastric and small intestinal conditions of newborn infants; however, recombinant Epo added to D(5)W was not protected from digestion. We conclude that Epo concentrations in milk increase as a function of the duration of breast feeding, that Epo is actively secreted into human milk by mammary duct epithelia, and that the Epo within milk is largely protected from digestion.