Lactose is the major osmotic agent in milk. Therefore, lactose synthesis indirectly regulates milk volume. The aim of this study was to determine the source of glucose and galactose in lactose. Six healthy lactating women were studied twice, during a 24-h fast and during ingestion of a mixed macronutrient drink (Sustacal) using [U-13C]glucose and [2-13C]glycerol. Six additional lactating women were studied on one single occasion during ingestion of glucose labeled with [1-13C]glucose. Using the ratios of [13C6] enrichments of glucose in lactose and plasma glucose and that of galactose in lactose and plasma glucose, we determined that 98 +/- 3% of glucose and 68 +/- 7% of galactose in lactose were derived from plasma glucose in the fed state, and 72 +/- 4 and 51 +/- 3%, respectively, after a 24-h fast. Virtually identical results (97 +/- 6 and 64 +/- 4%, respectively) were obtained during the glucose feeding study. On the basis of the [13C1] enrichment of glucose and galactose in lactose (derived from [2-13C]glycerol), glycerol contributes to the production of galactose but not glucose within the breast. Thus, plasma glucose is an important source of lactose, but significant amounts of glucose and galactose in lactose are generated within the breast, a process denoted hexoneogenesis. In this process, glycerol is a precursor for milk galactose but not glucose.