Characterization of the Pregnancy-Associated Glycoproteins (PAG) is important for studies of reproduction of various eutherian domestic, wild and endangered mammals. Distinct chorionic PAG genes are expressed in embryo-origin cells: pre-placental trophoblast (TR) and in placental trophectoderm (TRD) of various entherians. This study demonstrates in vitro production of the PAG proteins during long-term cultures of various chorionic explants: porcine TR or TRD, cotyledonary (CT) of European bison (Eb), and CT or intercotyledonary (intCT)-TRD of the cattle. Chorionic proteins isolated from media were analyzed by homologous or heterologous Western immunoblotting with anti-PAG sera, raised against cellular bovine or secretory porcine antigens. Used anti-PAG sera identified diverse molecular forms of released PAG proteins: 43-69 kDa for EbPAG proteins, 40-85 kDa for bovine PAG (bPAG), and 43-73 kDa for porcine PAG (pPAG). Immunoblotting revealed also that both CT and intCT-TRD explants secreted equivalent amounts of bPAG proteins. This useful system of in vitro protein production can provide native chorionic PAG proteins with placental unique carbohydrate chains. The PAG proteins are required as standard markers for diagnostic tests of pregnancy in domestic and wild mammals, in which seasonal reproductive processes are relatively difficult to control.