Antiserum directed against equine intestinal Alkaline Phosphatase (ALP) was produced in rabbits and used to develop a sensitive and quantitative assay for the detection of intestinal ALP in equine serum. This assay was then used to measure the half-life of intravenously injected intestinal ALP and to determine if the intestinal ALP was present in normal horse sera, sera from horses presented for lesions not involving the gastrointestinal tract and sera from horses presented with lesions involving the gastrointestinal tract. The results suggest that intestinal ALP is not likely to appear in equine serum even when gastrointestinal disease is present and, therefore, appears to be of no diagnostic value.