Metabolite levels in rat and cow mammary glands prepartum and at different stages of lactation were investigated, with a view toward determining whether changes in availabilities of energy sources and milk precursors to the mammary glands and changes in mammary metabolic regulatory systems are involved in effecting the initiation of lactation and milk yield in these species. Levels of energy substrates and milk precursors in the mammary glands of both species appeared adequate prepartum to support lactation. These observations were considered consistent with previous suggestions that the changes in cellularity and enzyme activities that occur after parturition in the rat might be responsible for initiation of lactation. Since no significant prepartum and postpartum differences in enzyme activities or availabilities of metabolites to the mammary gland have been observed in cows, the mechanisms involved in regulating the initiation of lactation in this species remain unresolved. The metabolite data presented suggest that the hexokinase and P-fructokinase reactions are rate-limiting in rat and cow mammary glands and represent potential regulatory sites in energy metabolism, as has been suggested for other tissues. © 1969, American Dairy Science Association. All rights reserved.