Neuropeptide Y (NPY) is abundant in plasma and amniotic fluid of women throughout pregnancy, during which its involvement in placental hormonogenesis has been proposed. In accordance with its putative role, the aim of this study was to characterize the human placental syncytiotrophoblast receptivity to NPY. Thus we performed this study on brush-border membranes (BBM) and basal plasma membranes (BPM). Specific ¹²⁵I-labeled NPY (¹²⁵I-NPY) binding to BBM was rapid (20 min), saturable, with a maximum binding capacity of 604 ± 100 fmol/mg protein, and of high affinity, with a dissociation constant of 11 ± 3 nM. No saturable binding could be shown in BPM. The rank order of affinity of NPY and related peptides to compete for ¹²⁵I-NPY binding sites was peptide YY (PYY) > NPY = [Leu³¹,Pro³⁴]NPY > _13-36NPY >> pancreatic polypeptide (PP). It is noteworthy that PYY displaced only 45% of the binding sites. In BBM, both NPY and PYY were potent phospholipase C (PLC) stimulators, leading to a four- to fivefold increase of control phosphodiesterase activity. The latter effect could be prevented by preincubation of membranes with 5 µM U-73122, a known inhibitor of G protein-linked receptor activation of PLC-. Furthermore, 5 µM BIBP-3226, a Y₁-receptor antagonist, shifted both dose-response curves to the right in a similar fashion for both peptides. In accordance with the PLC stimulation, both peptides also induced stimulation of protein kinase C (PKC) activity, which could be partially but additively prevented by U-73122 and LY-294002, a selective inhibitor of phosphatidylinositol-3 kinase (PI3K). Taken together, these data suggest that placental and blood-derived NPY binds to a mixed population of receptors composed of Y₁ and Y₃ subtypes on the maternal side of the syncytiotrophoblast, where it can mediate its physiological purposes via PLC- and PI3K activation, both of which lead to PKC activation. However, because BIBP-3226 antagonized both effects, the physiological relevance of the apparent Y₃ fraction is still unsolved.