To understand how changes in availability of glucose may influence milk production, two separate experiments were conducted in which the availability of glucose was either increased or decreased for lactating Holstein cows;In the first experiment, glucose availability was decreased and changes in concentrations of key energy metabolites were measured in five cows in early lactation and in negative energy balance. To cause glucosuria, phlorizin was injected subcutaneously every 6-h for 48 h at 0, 2, and 4 g/d. Blood was collected hourly from a jugular vein for the first and last 12 h of the 48-h period. Phlorizin caused 0, 225, and 337 g of glucose to be excreted into the urine daily. No effect was seen on milk production, but percentage of milk fat increased linearly with increasing phlorizin. Concentrations of glucose and insulin in plasma decreased linearly whereas concentrations of [beta]-hydroxybutyrate and nonesterified fatty acids increased linearly with increasing phlorizin. Diurnal variation, relative to feeding time, was seen for all metabolites measured. Results suggest that healthy, early-lactation cows in negative energy balance have the metabolic capacity to change substrates used for energy and for milk synthesis in order to compensate for short-term decreases in the availability of glucose;In the second experiment, four cows were used to quantify changes in the metabolism of rumen propionate, plasma glucose, and blood CO[subscript]2 when glucose was infused continuously into peripheral blood at 0, 342, or 737 g/d for at least 5 days. Neither production nor composition of milk was changed by the infusion of glucose. Irreversible losses of rumen propionate and blood CO[subscript]2 were not changed. Glucose treatments increased irreversible loss of glucose over the control in proportion to the amount of glucose infused, but did not change endogenous production of glucose. For the control treatment, 52% of plasma glucose was derived from propionate, with an additional 26% coming from other gluconeogenic sources. For the glucose treatments, the exogenous glucose was probably deposited in the body as energy reserves.