This study presents biochemical, histochemical, morphological and immunological evidence that part of the high molecular weight alkaline phosphatase observed in the serum of patients with liver disease and particularly in cases of intrahepatic cholestasis or focal-, extrahepatic obstruction originates from the liver plasma membrane. The high molecular weight protein alkaline phosphatase complex contains several plasma membrane enzymes and behaves like a plasma membrane fragment after isopycnic density gradient ultracentrifugation in sucrose, cesium chloride and metrizamide. Electron microscopic examination revealed a triple-layered vesicle which retained alkaline phosphatase activity. Incubation of human liver cells with anti-serum against purified high molecular weight multienzyme complex resulted in fixation of antibodies on the plasma membrane as shown by positive plasma membrane fluorescence. These plasma membrane fragments in the serum are not of biliary origin.