ABSTRACT: The antigens recognized by heteroantisera raised to human trophoblast membrane were studied on a variety of normal tissues and certain neoplastic cell lines, both by immunohistological and immunoprecipitation techniques. Native antisera reacted with all tissues examined, but after absorption with normal human serum and lyophilized normal liver, reactivity with normal tissues was restricted to the trophoblast membrane, endocervix, and mitogen- and antigen-stimulated lymphocytes. Several membrane components were precipitated from trophoblast and activated lymphocytes by native antisera, whereas after absorption a single radioactive peak of MW 62,000 was obtained. HeLa, Chang, AV3, and HEp-2 cell lines were also positive by immunofluorescence with the absorbed antisera, and a single molecular species was again precipitated. Additional enzymatic studies both of immunoprecipitated material and of the tissue of origin provided evidence that this species was the placental isoenzyme of alkaline phosphatase. These results indicate that the major specificity in absorbed trophoblast antisera is the heat-stable placental isoenzyme of alkaline phosphatase. Furthermore, since similar material appears to be present on other normal and transformed tissues, this isoenzyme may not therefore, despite previous claims, be truly trophoblast-specific.