Alkaline phosphatase isoenzymes of equine tissues, peritoneal fluid, and serum were characterized by their electrophoretic mobilities, using polyacrylamide gel disc electrophoresis. The alkaline phosphatase isoenzymes in liver, kidney, spleen, small intestine, placenta, bone, small colon, and large colon tissue samples were extracted and separated by electrophoresis. The resulting isoenzyme mobilities and spectrophotometric scans were evaluated for their tissue specificity and for their possible use in determining the tissue contribution of alkaline phosphatase to serum and peritoneal fluid. The sensitivity of the tissue alkaline phosphatase to heating at 56 C was also determined. The isoenzymes of all the tissue extracts, except small intestine and large colon, were distinguishable from one another by their specific electrophoretic mobilities and patterns and by their sensitivity to heating at 56 C. The mobilities and patterns of small intestine and large colon isoenzyme extracts were not different enough to allow making a differentiation, but the heat resistance of small intestine did distinguish it from large colon. The tissue sources of alkaline phosphatase in the serum of healthy adult horses, pregnant mares, and normal foals and in the peritoneal fluid of healthy horses were determined by characterization of the mobilities of their isoenzymes.