Phosphorylation of human vescicle docking protein p115 at Ser-942 (homologous to Ser-940 in rat p115) promotes its dissociation from the Golgi membrane, Here we show that a peptide encompassing the 934-950 sequence of p115 is unaffected or poorly phosphorylated by a variety of Ser/Thr protein kinases with the notable exception of the Golgi apparatus casein kinase (G-CK) which phosphorylates it with an efficiency comparable to that of its optimal peptide substrates. In contrast phosphorylation of the p115 peptide by protein kinase CK2 is negligible compared to that of the specific peptide substrates of this kinase, Phosphorylation by G-CK is abolished if a conserved cluster of acidic residues at position between n + 4 and n + 9 (EDDDDE) is replaced by a neutral stretch (GAG;AG;A). These data strongly support the view that G-CK but not the other two classes of ubiquitous "casein kinases" (CK1 and CK2) is the natural phosphorylating agent of p115.