Regulator of ubiquitous kinase/Cbl-interacting protein of 85 kDa (Ruk/CIN85) and CD2-associated protein/Cas ligand with multiple SH3 domains (CD2AP/CMS) comprise a family of vertebrate adaptor proteins involved in several important cellular processes, including downregulation of activated receptor tyrosine kinases, regulation of cytoskeletal rearrangements, phosphatidylinositol 3-kinase (PI 3-kinase) signalling and apoptosis. The role of Ruk/CIN85 as a scaffold protein involved in membrane trafficking processes has been demonstrated in model cell systems. However, intracellular localization of endogenous Ruk/CIN85 has never been comprehensively assessed. We carried out detailed studies of subcellular distribution of Ruk/CIN85 in adherent cultured human cells using antibodies that recognize distinct epitopes of the protein and revealed a punctate immunostaining pattern, common for proteins involved in intracellular trafficking processes. Our data indicate that Ruk/CIN85 is distributed between several different membrane trafficking compartments, but the major pool of Ruk/CIN85 is associated with the Golgi complex, mainly with a subpopulation of COPI-coated vesicles involved in retrograde endoplasmic reticulum-Golgi and intra-Golgi transport. This localization pattern is dependent on the integrity of Golgi complex and intact microtubular network. Only a small pool of Ruk/CIN85 is present in compartments involved in clathrin-mediated endocytosis and sorting. These results suggest that endogenous Ruk/CIN85 may be involved in regulation of specific membrane trafficking processes.