In budding yeast, over 60 proteins functioning in at least five modules are recruited to endocytic sites with predictable order and timing. However, how sites of clathrin-mediated endocytosis are initiated and stabilized is not well understood. Here, the casein kinase 1 (CK1) Hrr25 is shown to be an endocytic protein and to be among the earliest proteins to appear at endocytic sites. Hrr25 absence or overexpression decreases or increases the rate of endocytic site initiation, respectively. Ede1, an early endocytic Eps15-like protein important for endocytic initiation, is an Hrr25 target and is required for Hrr25 recruitment to endocytic sites. Hrr25 phosphorylation of Ede1 is required for Hrr25-Ede1 interaction and promotes efficient initiation of endocytic sites. These observations indicate that Hrr25 kinase and Ede1 cooperate to initiate and stabilize endocytic sites. Analysis of the mammalian homologs CK1δ/ε suggests a conserved role for these protein kinases in endocytic site initiation and stabilization.