Blood, serum, and plasma total calcium concentrations and plasma and serum ionized calcium concentrations were anaerobically determined by use of a calcium-specific electrode for samples obtained from 39 healthy horses. Mean (+/- SD) serum ionized calcium concentration was 6.6 +/- 0.3 mg/dl (1.6 +/- 0.1 mmol/L) and the mean serum ionized calcium percentage was 58.2 +/- 3.4%. Serum ionized calcium percentage was not significantly correlated with serum pH. Plasma ionized calcium percentage was weakly correlated with plasma pH (r = - 0.480; P less than or equal to 0.05). Ionized calcium concentration was determined in serum samples manipulated in vitro by additions of 1 to 80 microliter of 0.1N hydrochloric acid or sodium hydroxide to yield 6 to 10 pH values between 6.8 and 8.2. In all horses, the relationship between serum ionized calcium percentage and serum pH at these pH values was then examined by use of a repeated-measures multiple regression analysis. Correlations between serum ionized calcium percentage and adjusted serum pH value for each horse were highly significant (P < 0.05); however, analysis of pooled data from all horses indicated that a statistically significant relationship between serum pH and ionized calcium percentage did not exist. Lack of a significant relationship between these variables was most likely attributable to heterogeneity of variance of ionized calcium percentage among horses, reflecting variation in undefined biochemical constituents of serum that affect the equilibrium of calcium binding. When it is essential to evaluate the calcium status of a horse, direct measurement of serum ionized calcium concentration is recommended.