NELL proteins are the thrombospondin-1-like proteins that are strongly expressed in neural tissues, containing six epidermal growth factor (EGF)-like domains. By radiolabeling of the NELL protein-expressing COS-7 cells with [³²P]orthophosphate, here we demonstrate that NELL proteins are synthesized as phosphoproteins by interacting with protein kinases in the cells. By immunoprecipitation and in vitro phosphorylation assays, we have also found that NELL proteins expressed in COS-7 cells are associated with and phosphorylated by protein kinase C βI (PKCβI). Further analysis using various deletion mutants of NELL proteins by the yeast two-hybrid assay has revealed their EGF-like domains to be involved in the isoform-specific interaction with PKC. Conversely, the NH₂-terminal variable region of PKC isoforms has been found essential for the interaction with NELL proteins. Because NELL proteins are expressed mainly in the cytoplasm of neuronal cells, unlike most EGF-like domain-containing extracellular proteins, the novel protein–protein interaction identified here between the EGF-like domains of NELL proteins and PKC suggests that EGF-like domains of intracellular proteins can be a target of PKC that mediates various signaling pathways.