Twelve lactating Holstein cows were blocked according to age and milk production into groups of three cows and assigned to three 4 x 4 Latin squares in a split-plot design with subtreatments. Treatments on each square were four diets formulated to provide -10, 0, +10, or +20 meq/Na + K) -Cl/100 g diet DM. The four balances were achieved on squares 1, 2, and 3 by manipulating Na, K, and Cl, respectively. Actual milk yield was 8.6% higher on +20 than -10 averaged across the three squares. Blood pH and bicarbonate increased linearly with dietary cation-anion balance. Rumen pH increased linearly with dietary cation-anion balance, but fermentation patterns were largely unaffected. Urine pH increased linearly and quadratically with increasing dietary cation-anion balance. Square times balance response differences proved nonsignificant for all parameters except blood bicarbonate and rumen isovalerate, indicating responses could be attributed to the dietary cation-anion balance itself rather than to the effects of a single ion. Regulation of dietary cation-anion balance may become a useful tool for improving the performance of lactating dairy cattle.