This new method for enzymatic assay of serum chloride is based on determination of chloride-dependent alpha-amylase (EC 3.2.1.1) activity. We evaluated the ion specificity and practicability of the method for routine use with the Hitachi 705. The analytical range of the method extends from 40 to 160 mmol of chloride ion per liter of serum. The reaction rate for samples containing 100 mmol of chloride per liter was 0.17 A/min. CVs within-run and between-run were less than 1.0%. Correlation with results of a coulometric titration method was good. The specificity for bromide is 75% of that for chloride; for the other anions, it is 0%. This enzymatic method is generally applicable to wide variety of automated chemistry analyzers.