Aim of the study: This study aimed to elucidate mechanisms that rearrange endosomal system in the early phase of MCMV infection. Therefore, we investigated the function of cellular proteases and major endosomal routes in infected cells as well the MCMV influence in the intracellular expression levels of regulatory protein families Rab and Arf. Materials and methods: For our experiments, we used murine embryonic fibroblasts (MEF) and immortalized fibroblast-like Balb3T3 cells and two recombinant MCMV viruses: Δm138-MCMV and Δ9-MCMV. Intracellular distribution and expression of endosomal regulatory proteins, endosomal marker proteins, and viral proteins, was analyzed by immunofluorescence techniques and imaging by confocal microscopy. Cellular kinetic parameters were determined by flow cytometry (using in-house developed software, made in collaboration with Faculty of Engineering of the University of Rijeka). The intracellular expression levels of endosomal markers and viral proteins were quantified by Western blot. Molecules were followed with or without the application of different chemical inhibitors. Results: Functional system of cellular proteases that are active in the neutral pH environment (including furin), is required for the development of MCMV infection. Functional assays revealed alteration of the endosomal flow in the early phase of MCMV infection, as demonstrated by several cargo molecules, including trafficking of TfR, MHC-I molecules and NBD-sphingomyelin through the cellular endosomal system. Their recycling was delayed, and cargo molecules were retained in EPERC, the perinuclear endosomal compartment remodeled by MCMV. The kinetic modeling revealed that MCMV infection does not affect the rapid recycling route, but inhibits the egress from sorting endosomes (SE) and endosomal recycling compartment (ERC) as well as transfer form SE to ERC. The inhibition of the egress from SEs and the ERC was associated with altered levels and subcellular distribution of cellular regulatory proteins, most importantly Arf6, Rab35, and Rab22A (demonstrated by the immunofluorescence imaging and Western blot analysis). Conclusion: Serine proteases (i.e. furin) are necessary for the regular development of infection. MCMV infection causes retention of endocytosed cargo in SEs and the ERC which is associated by redistribution of the small GTPases from Rab and Arf families (Arf6, Rab35, and Rab22A.