1. The incorporation of radiolabelled plasma glucose into mammary lactose was used to measure the rate of lactose synthesis in lightly anaesthetized lactating rats. 2. Lactose synthesis showed a diurnal variation with a minimum at 18:00h 3. Food withdrawal for 6h did not affect lactose synthesis in the early morning but greatly decreased it in the afternoon or evening. 4. Plasma glucose, milk sugars and total galactosyltransferase activity (EC 2.4.1.22) did not show the above changes. 5. Measurements of plasma insulin, which varies diurnally, and experiments with injected insulin suggested that variations of insulin within the physiological range do not account for the changes in lactose synthesis described.