Human alpha-amylase (alpha-1,4-glucan-4-glucanohydrolase, EC 3.2.1.1) was separated by electrofocusing. The amylases in serum and urine were seperated into two major isozymes with isoelectric points of pH 6.4-6.5 and pH 6.9-7.0, respectively, and one minor isozyme with an isoelectric point of pH 5.9. The amylases in saliva were separated into one major isozyme with an isoelectric point of pH 6.5 and two minor isozymes with isoelectric points of pH 6.0 and 6.9. The amylases in pancreatic juice gave one major peak at pH 7.0 and two minor peaks at pH 6.0 and pH 6.5. Repeated electrofocusing of each isozyme have only a single peak with a constant isoelectric point. The antigenicites of these three isozymes were also investigated using antisera to rat pancreatic and parotid amylases.