We describe a new method for separating alkaline phosphatase isoenzymes by "high performance" liquid chromatography. Isoenzymes are chromatographed on the column (Mono Q HR 5/5, a strong anion-exchanger) with stepwise gradient elution. The isoenzymes originating from small intestine, bone, liver and bile were identified by use of tissue homogenates, pathological sera, and heat inactivation. One isoenzyme was identified in the homogenate of small intestine, two were identified in bone, and two in liver, and biliary and fragment isoenzymes in bile. There are indications that the first liver isoenzyme is derived from the cell membrane and the second liver isoenzyme from the cytosol. The biliary isoenzyme is considered to be a highly sensitive and specific indicator for cholestasis. We propose that the isoenzymes called "Fragmented Isoenzymes" which had been detected in liver homogenate and in bile fluid, represent catabolites of the various isoenzymes in serum.