This paper is a study to identify the clinical significance of high-molecular-mass alkaline phosphatase (ALP:E:C..3.1.3.1.), ALP-lipoprotein-X complex (LP-X) and intestinal variant ALP. We used cellulose acetate and agarose gels and techniques including wheat germ lectin, cetavlon-diethyl ether, thermostatability, neuraminidase and L-phenylalanine to improve the electrophoretic separation of the alkaline phosphatase isoenzymes. Patients' serum samples were electrophoresed from a diverse group of individuals ill with cholestasis, neoplastic disease metastatic to the liver, hepatocellular carcinoma, cirrhosis, diabetes mellitus, and chronic renal disease. Agarose gels provided better separation of ALP isoenzymes than cellulose acetate gels. The results also indicated that high-molecular mass ALP is present in patient's serum in conditions associated with cholestasis especially caused by hepatic malignancy. High-molecular mass ALP was frequently found to co-exist with the liver isoenzyme and LP-XALP complex. The intestinal variant was identified in patients with malignancy, cirrhosis, chronic renal disease and diabetes mellitus. Intestinal ALP coexisted concomitantly with a variant intestinal ALP. Intestinal variant ALP is most likely composed of intestinal ALP attached to a cellular membrane-binding domain, or may be an artifact produced by neuraminidase incubation.