Eighty multiparous Holstein cows were assigned to eight treatments in a 2 x 2 x 2 factorial design to examine changes in serum parathyroid hormone, calcitonin, Ca, P, Mg, K, and Cl under two levels of dietary Ca and P with two anion-cation balances. Factor levels were low and high Ca (51 vs. 115 g/d), P (38 vs. 52 g/d), and cationic:anionic balance (23 vs. -8 meq). Cows were offered a TMR and an experimental mineral supplement to adjust mineral and anion-cation levels. Caudal vein blood samples were collected every 2 d from d -10 to +10 from calving. Serum K was lower for low Ca and high P compared with high Ca and low P treatments. Neither hormones nor the minerals examined in serum showed treatment effects. Cows of higher parity consumed less supplement and had lower serum Ca and P. All serum variables except calcitonin showed day to day variations. Both Ca and P decreased around parturition, whereas parathyroid hormone and Mg increased. Anionic diets did not differ from cationic diets regarding serum parathyroid hormone, calcitonin, Ca, P, Mg, K, Cl, or Na.