The Delta/Serrate/LAG-2 (DSL) domain-containing proteins, Delta1, Jagged1, and Jagged2, are considered to be ligands for Notch receptors, However, the physical interaction between the three DSL proteins and respective Notch receptors remained largely unknown. In this study, we investigated this issue through the targeting of Notch1 and Notch3 in two experimental systems using fusion proteins comprising their extracellular portions. Cell-binding assays showed that soluble forms of Notch1 and Notch3 proteins physically bound to the three DSL proteins on the cell surface, In solid-phase binding assays using immobilized soluble Notch1 and Notch3 proteins, it was revealed that each DSL protein directly bound to the soluble Notch proteins with different affinities. All interactions between the DSL proteins and soluble Notch proteins were dependent on Ca2+. Taken together, these results suggest that Delta1, Jagged1, and Jagged2 are ligands for Notch1 and Notch3 receptors,