Three types of white perch (Morone americana) vitellogenin (VtgAa, VtgAb, and VtgC) were purified, labeled with digoxigenin (DIG), and subjected to Vtg receptor (Vtgr) binding assays in 96-well plates coated with perch ovarian membrane proteins or to ligand blotting procedures. Binding specificity was evaluated by incubating membrane protein preparations with constant amounts of DIG-Vtg tracer (VtgAa, VtgAb, VtgC, or a mixture of VtgAa and VtgAb [VtgAa/b]) alone or in the presence of unlabeled Vtg ligands. At 250-fold excess molar concentration relative to the tracer, VtgAa and VtgAb were each able to displace only approximately 50% of bound DIG-VtgAa/b, but VtgAa/b could fully displace DIG-VtgAa and DIG-VtgAb under the same conditions. Over a broad range of excess molar ratios, unlabeled VtgAa and VtgAb each displaced their respective DIG-Vtg tracer much more effectively than each did the heterologous tracer (DIG-VtgAb and DIG-VtgAa, respectively). Ligand blotting revealed three forms of Vtgr, a large receptor (>212 kDa) that bound only to VtgAa and two smaller receptors (∼ 116 and ∼ 110.5 kDa) that bound preferentially to VtgAb. The VtgC did not specifically bind to ovarian membrane proteins in either assay. Collectively, these results indicate the presence of a system of multiple ovarian Vtgrs with disparate binding to the three types of Vtg present in higher-order teleosts (Acanthomorpha). To our knowledge, this is the first report on binding of multiple types of Vtg to multiple forms of Vtgr in any vertebrate.