The separation and quantitation of the various human serum alkaline phosphatases has been largely by indirect and/or imprecise methods. A rocket electroimmunoassay method has been developed for the quantitation of the alkaline phosphatases, which uses enzymatic activity to detect the rocket and bromochloroindolylphosphate as substrate. The assay for intestinal phosphatase requires only one-dimensional electrophoresis, since the antibody to the intestinal enzyme does not cross-react with the other phosphatases. Both liver and bone enzymes give a line of identity when tested against antibody directed against the liver phosphatases, and these phosphatases require separation first by acrylamide gel electrophoresis before quantitation by crossed immunoelectrophoresis. Liver/bone enzyme levels in serum are easily detected within the linear range of the assay without concentration of the serum. The assay is 10-fold more sensitive than quantitation by acrylamide gel electrophoresis. The inability to detect circulating intestinal alkaline phosphatase in the serum of most fasting hospitalized patients has been documented by this sensitive assay, and the predominance of the liver isoenzyme has been confirmed. The assay should prove useful for determining the tissue of origin of serum alkaline phosphatases, and in providing quantitative data for physiological studies. It is not adaptable for automation and will not prove useful in the routine clinical laboratory.