We studied effects of breed, dietary fat source, and dietary copper intake as factors known to influence unsaturation of milk fat and its potential for development of spontaneous oxidized flavor in milk. Twelve Holstein and 12 Jersey cows were allotted to three blocks with four cows of each breed. Cows within breed were allotted randomly within blocks and fed control or copper-depleting diets for 2 mo to achieve stable or depleted liver copper stores. Cows then were fed tallow or roasted whole soybeans in a two-period switchback (5 wk per period); during the last week of each period additional vitamin E (2000 IU/d) was added. Copper depletion for 2 mo decreased concentrations of copper in liver. Feed intake and milk yield were influenced only by breed. The proportions of C4:0 to C14:0 and C18:0 in milk fat were higher, whereas C16:1 and cis-C18:1 were lower in Jersey cows. Feeding soybeans increased C4:0 to C14:0, C18:0, C18:2, and C18:3 in milk, and decreased C14:1, C16:0, C16:1, trans-C18:1, and cis-C18:1. Depleted copper status increased conjugated linoleic acid in milk. Several breed x fat source interactions for individual milk fatty acids occurred. Feeding soybeans decreased plasma concentrations of copper and zinc, and increased concentrations of alpha-tocopherol in plasma and milk. The concentration of zinc was higher in milk of Jersey cows. Depleted copper status tended to increase copper concentration in plasma and decreased copper in milk. Fat source did not influence plasma copper concentration when status was adequate, but plasma copper concentration was higher when tallow was fed to cows with depleted copper status. Supplementing vitamin E increased concentration of alpha-tocopherol in plasma and milk and decreased concentration of zinc in milk. Factors influencing the potential for oxidized flavor development in milk can be manipulated by changing the diet of the cow.