Compared to hay, when concentrates were fed three- to fourfold, increases occurred in the activities of enzymes associated with fatty acid synthesis and esterification in adipose tissue, whereas mammary enzyme levels were relatively unaffected. The proportion of stearic acid in milk fat, presumably arising from adipose tissue, was significantly depressed in the high response group (severe milk fat depression) but not in the low response group (moderate milk fat depression). Differences in dietary effects upon mammary intermediary metabolite levels between the high and low response groups suggested a relatively lower availability of energy to mammary glands of cows in the high response group when fed concentrate. A number of changes in adipose tissue metabolite levels were noted in concentrate-fed animals, the most interesting being a twofold increase in the level of α-glycerol-P in the high response group. These observations were consistent, with a previous suggestion that severe milk fat depression can be attributed, in part, to a decrease in the availability of long-chain fatty acids for milk synthesis. Alterations in mammary gland metabolism may occur, even though no significant enzymatic changes occur in mammary tissue when milk fat percentage is depressed. © 1969, American Dairy Science Association. All rights reserved.