Summary: Metabolites in the lactose synthesis pathway were measured in human milk during established lactation. The mean concentrations (range) were: lactose, 198 m M (175–233); glucose, 1.5 m M (0.1–2.4); glucose 6‐phosphate, 11 μ M (4–23); glucose 1‐phosphate, 2.7 μ M (1.6–6.8); UDP‐glucose, 2.7 μ M (0.6–6.0); UDP‐galactose, 0.7 μ M (<0.1–2.5); inorganic phosphate, 2.2 m M (0.8–2.9). During storage of milk within the breast for 2 h, glucose 6‐phosphate concentration increased by 1.4 ± 0.5 μ M, while UDP‐glucose decreased by 1.0 ± 0.4 μ M. None of the other metabolites showed significantchanges in concentration during this period. Only glucose showed a circadian rhythm in concentration, with the highest concentrations between 1400 and 1800 h. The volumes of milk produced by the mothers ranged from 178 to 1,739 ml/24 h. Despite this variation, there were no significant relationships between the 24‐h average concentrations of glucose, glucose 6‐phosphate, glucose 1‐phosphate, UDP‐glucose, UDP‐galactose, or inorganic phosphate and the amount of lactose produced over 24 h.