Successful pregnancy is dependent upon an array of signaling proteins secreted by the trophoblast cells of the placenta. Among these is a group of proteins related to pituitary prolactin, known as the prolactin/growth hormone family. These proteins are expressed at specific times during gestation and synthesized in distinct trophoblast cell types in the rat placenta. We report here that select members of this family, prolactin-like protein (PLP-A), PLP-B, PLP-C, decidual/trophoblast PRP, and placental lactogen I variant, only which are expressed in the spongiotrophoblast, late in rat placental development bear Asn-linked oligosaccharides terminating with NeuAc␣2,6GalNAc1,4GlcNAc-R. This reflects the concurrent expression of these prolactin/growth hormone family members with the peptide-specific 1,4Gal-NAc-transferase and an ␣2,6-sialyltransferase, which can add sialic acid to terminal 1,4-linked GalNAc. We have determined that at least one of the prolactin-like proteins, PLP-A, is recognized by the protein-specific GalNAc-transferase. The presence of NeuAc␣2,6GalNAc-1,4GlcNAc-R on only a limited number of glycoproteins synthesized by the spongiotrophoblasts between mid gestation and birth reflects the need for both the GalNAc-transferase and the peptide recognition determinant for efficient addition of GalNAc. Thus, expression of the GalNAc-transferase and specific members of the prolactin/growth hormone family is developmentally regulated in the rat placenta, suggesting a physiological role for the terminal NeuAc␣2,6GalNAc1,4Glc-NAc-R sequence on Asn-linked oligosaccharides of these proteins.The placenta is a complex organ that is essential for successful growth and maturation of the fetus throughout pregnancy (1). In addition to bringing oxygen and nutrients to the fetus, the placenta produces a number of hormones, cytokines, and growth factors which influence the endocrine, immune, and metabolic functions of the mother. Among these is a group of closely related proteins synthesized by the rat placenta, the prolactin/growth hormone family. We demonstrate here that specific members of this family bear asparagine (Asn)-linked oligosaccharides with NeuAc␣2,6GalNAc1,4GlcNAc. This is the result of high expression levels of a protein-specific GalNAc-transferase in the rat placenta. Levels of the transferase increase from undetectable at mid gestation to maximal at day 18 of gestation. This corresponds precisely with the expression of the prolactin/growth hormone family members PLP 1 -A, PLP-B, PLP-C, d/t PRP, and PL-Iv by spongiotrophoblasts from the junctional zone of the placenta. At least one of these family members, PLP-A, contains a recognition determinant that can be utilized by this protein-specific GalNAc-transferase, which we have previously shown (2-5) to mediate 1,4-linked GalNAc addition to Asn-linked oligosaccharides on the pituitary glycoprotein hormones lutropin and thyrotropin.We have shown previously that unique Asn-linked oligosaccharides terminating with the sequence SO 4 -4-GalNAc1, 4...