The alkaline phosphatase in homogenates of human liver was separated into two components by the addition of Triton X-100 to an agarose gel electrophoretic system. One of these components migrated at a rate identical to that of the original one and similar to alpha2-macroglobulin. The other component migrated more slowly, at a rate that resembled that of beta1-transferrin. Human serum samples regularly contained an identical fast-migrating fraction, whereas an identical slowly migrating fraction only appeared in serum obtained from patients with various diseases, especially from patients with malignant tumours, even though the liver did not contain tumour metastases. Slow isoenzyme was found in a few sera that had alkaline phosphatase activity within the normal range. Histochemical examinations of liver tissue from patients whose serum contained the slowly migrating isoenzyme showed a pronounced reaction of alkaline phosphatase in the bile canaliculi, and this isoenzyme seems to arise from the canaliculi. The fast-migrating isoenzyme might arise from the endothelial cells of the liver, to which the activity is usually confined in histochemical stainings.