We have previously demonstrated that plasma glucose contributed 80% in the fed and 60% in the fasted state to lactose synthesis in humans, and de novo synthesis in the breast contributing to both the glucose and galactose moieties accounted for the remaining 20% and 40%, respectively, of lactose. The present study was conducted to determine, in lactating women, whether oral galactose is directly incorporated from plasma galactose into glucose and galactose in milk lactose or via conversion of galactose to glucose in the liver. Six healthy exclusively breast-feeding women (30 +/- 2 yr) (mean +/- SE) ingested galactose at 22 micromol x kg-1 x min-1 for 9 h after an overnight fast during infusion of [6,6-2H2]glucose and [1-13C]galactose. We observed that 69 +/- 6% of glucose and 54 +/- 4% of galactose in lactose were derived directly from plasma glucose, whereas 7 +/- 2% and 12 +/- 2% of glucose and galactose in lactose, respectively, were derived directly from plasma galactose. De novo synthesis of glucose and galactose via hexoneogenesis accounted for 25 +/- 8% and 35 +/- 6%, respectively. We conclude that during ingestion of galactose the contribution from plasma glucose to glucose and galactose in lactose was similar to that of a short-term fasting, but part of the de novo synthesis of glucose and galactose in the breast was replaced by direct uptake of galactose.