OBJECTIVE To compare the anion gap (calculated as the sodium concentration minus the sum of the chloride and total carbon dioxide concentrations) reference interval for three automated chemistry analyzers. METHODS We measured serum sodium, chloride, and total carbon dioxide on aliquoted specimens using three commercial instruments. Quality control and proficiency survey materials were run to ensure that the analyzers were functioning optimally. METHODS Three separate clinical laboratories affiliated with one university medical center participated in the study. METHODS Healthy volunteers from 20 to 60 years of age were recruited from within a clinical laboratory. METHODS The mean and standard deviations of the anion gaps measured by each method were calculated. RESULTS The parametric reference intervals (+/-2 SD from the mean) were 5 to 10 mmol/L for the Beckman Synchron CX3 analyzer, 9 to 14 mmol/L for the Boehringer Mannheim Hitachi 717 analyzer, and 8 to 13 mmol/L for the Johnson & Johnson Vitros 950 analyzer. CONCLUSIONS Our results suggest that while it may be appropriate to lower the anion gap reference interval to 5 to 10 mmol/L for some analyzers, as suggested by earlier reports, 9 to 14 mmol/L may be a more appropriate reference interval for other analyzers. For the anion gap to be an effective tool for diagnosing acid-base disorders, clinical laboratorians need to establish (or at least verify) the anion gap reference interval for the instrumentation used in their laboratory, inform clinicians of this reference interval, and perform quality control studies to ensure that the reference interval for this calculated result remains valid.