Activities of nine enzymes and the levels of 15 metabolites were determined in mammary glands from normal (N) and adrenalectomized–ovariectomized (AO) rats in midlactation. Mammary gland weights and mammary DNA did not change between day 11 and day 19 of lactation in N animals and were not affected appreciably by AO. Four days after AO the activities of citrate cleavage enzyme, malic enzyme, UDPglucose pyrophosphorylase, the A protein of the lactose synthetase complex, and glucose-6-phosphate dehydrogenase were decreased to 24, 59, 53, 52, and 56% of day 11 N. These same enzymes require cortisol therapy to increase in activity after adrenalectomy on the 5th day of lactation. Activities of phosphoglucomutase, 6-phosphogluconate dehydrogenase, fructose-1,6-diphosphate aldolase, and isocitrate dehydrogenase were not affected by AO. These enzymatic changes are discussed in relation to systems regulating milk synthesis. Mammary glucose levels increased 1.9-fold in AO animals. Levels of triose phosphate, α-glycerol phosphate, and aspartate in AO animals were decreased to 52, 49, and 62% of N. The proportion of high-energy phosphate bonds to total adenine nucleotides was not affected by AO. Ratios of [free NADP]/[free NADPH2] were not significantly affected by AO or stage of lactation. Metabolite patterns are discussed in relation to changes in enzyme activities and in vitro tracer data.