Transport through the endocytic pathway is inhibited during mitosis. The mechanism responsible for this inhibition is not understood. Rab4 might be one of the proteins involved as it regulates transport through early endosomes, is phosphorylated by p34(cdc2) kinase, and is translocated from early endosomes to the cytoplasm during mitosis. We investigated the perturbation of the rab4 GTPase cycle during mitosis. Newly synthesized rab4 was less efficiently targeted to membranes during mitosis. By subcellular fractionation of mitotic cells, we found a large increase of cytosolic rab4 in the active GTP-form, an increase not associated with the cytosolic rabGDP chaperone GDI. Instead, phosphorylated rab4 is in a complex with the peptidyl-prolyl isomerase Pin1 during mitosis, but not during interphase. Our results show that less efficient recruitment of rab4 to membranes and a bypass of the normal GDI-mediated retrieval of rab4GDP from early endosomes reduce the amount of rab4GTP on membranes during mitosis. We propose that phosphorylation of rab4 inhibits both the recruitment of rab4 effector proteins to early endosomes and the docking of rab4-containing transport vesicles. This mechanism might contribute to the inhibition of endocytic membrane transport during mitosis.