• We examined the effects of variations of serum phosphate levels on serum ionized calcium concentrations in vitro. A single donor serum sample was divided into 25 aliquots stored in tubes sealed with carbon dioxide and divided into 5 subsets of tubes. The pH was altered in 4 of the 5 subsets by adding various concentrations of hydrochloric acid or sodium hydroxide. The pH levels studied ranged from 7.09 to 7.63. The phosphate concentration was altered in each subset by adding various concentrations of a phosphate buffer. The phosphate concentrations studied ranged between 0.81 and 3.58 mmol/L. There was an inverse relationship between ionized calcium and phosphate at all pH levels studied. The ionized calcium concentration correlated inversely with pH. We suggest that in addition to factors well known to influence serum ionized calcium concentration (such as protein, bicarbonate, and pH values), serum phosphate concentration also plays an important role.