No non-invasive methods exist to accurately evaluate Cu, Zn, and Mn status in dairy cows. Thirty lactating Holstein cows were used to determine whether intake and source of Cu, Zn, and Mn affected erythrocyte Cu/Zn SOD activity and expression of genes in milk fat that are related to Cu and Zn transport. Cows were fed a diet void of supplemental Cu, Zn, and Mn (9, 41, and 41 mg/kg, respectively) for 30 d and then fed 1 of 3 diets for 30 d. One diet (UNSUP) contained no supplemental Cu, Zn, and Mn (9, 41, and 41 mg/kg); one diet (SUL) contained Cu, Zn, and Mn from sulfates (total concentrations = 17, 59, and 54 mg/kg, respectively); one diet (GLY) contained Cu, Zn, and Mn in the glycinate form (B-Traxim 2C, Pancosma; total concentrations = 20, 66, and 58 mg/kg). Data were analyzed using a mixed model with treatment and parity as fixed effects. Using the NRC (2001) model and absorption coefficients (AC), UNSUP provided about 82% of requirements for Cu and Zn for primiparous cows and 95% for multiparous cows. Assuming an AC of 0.05 and 0.20 for Cu and Zn from supplements, supplemented diets provided 1.3 to 2.3 times more absorbed Cu and Zn than requirements. Total RNA was extracted using Tri Reagent from milkfat samples collected on d 60. Expression of 1 Zn transport gene (ZnT4), 2 Zn and Mn transport genes (Zip3 and Zip8), 2 Cu transport genes (ATP7B and CCS), 1 heavy-metal binding gene (MT), and 2 reference genes (KEAP1 and ADSL) were analyzed using qPCR. Activity of SOD was higher (P = 0.04) in primiparous than multiparous cows, but was not affected by diet. Expression of CCS, a protein that transports Cu to SOD, and SOD were negatively correlated (P < 0.06). Expression of most genes was not affected by treatment; however, MT expression was greater (P = 0.04) in primiparous compared with multiparous cows, and Zip8 expression tended (P = 0.10) to be greater in cows fed supplemental Cu, Zn, and Mn regardless of source. When supplemental Cu, Zn, and Mn were fed, cow requirements were exceeded and no differences were observed between mineral sources. Zip8 has potential to detect cows with deficient trace mineral status.