The Golgi apparatus is a dynamic membranous structure, which has been observed to alter its location and morphology during the cell cycle and after microtubule disruption. These dynamics are believed to be supported by a close structural interaction of the Golgi with the microtubule cytoskeleton and associated motor enzymes. One microtubule-dependent motor enzyme, kinesin, has been implicated in Golgi movement and function although direct evidence supporting this interaction is lacking. In this study, we utilized two well-characterized kinesin antibodies in conjunction with subcellular fractionation techniques, immunoblot analysis and immunofluorescence microscopy to conduct a detailed study on the association of kinesin with the Golgi and other membranous organelles in a polarized epithelial cell, the primary rat hepatocyte. We found that kinesin represents approximately 0.3% of total protein in rat liver homogenates, with approximately 30% membrane-associated and the remainder in the cytosol. Among membrane fractions, kinesin was concentrated markedly in Golgi-enriched fractions, which were prepared using two independent techniques. Kinesin was also abundant in fractions enriched in transcytotic carriers and secretory vesicles, with lower levels detected on fractions enriched in endosomes, endoplasmic reticulum, lysosomes and mitochondria. Immunofluorescence microscopy showed that kinesin is concentrated on Golgi-like structures in both primary cultured hepatocytes and rat hepatocyte-derived clone 9 cells. Double-label immunofluorescence demonstrated that kinesin staining colocalizes with the Golgi marker, alpha-mannosidase II, in both cell types. These results provide compelling evidence showing that kinesin is associated with the Golgi complex in cells and implicate this motor enzyme in Golgi structure, function and dynamics.