Cytomegaloviruses (CMV) are a double-stranded DNA virus that belong to the beta herpes virus family. All beta-herpesviruses have the property of reorganizing the cell membrane system to create an environment that is favorable for their own replication. Reorganization of cell compartments and membranes ultimately results in the creation of a cytoplasmic virion assembly compartment - cVAC. The aim of this study was to examine whether the expression and intracellular localization of Arf GEF regulatory proteins Gbf1, Big1 and Big2 change during CMV infection and to examine whether their impaired function by small inhibitors impairs establishment of cytomegalovirus infection. Western blot has shown that expression of Big1, Big2 and Gbf1 increses during CMV infection and Big1 expression is highest after 6 hours of infection, while Big2 and Gbf1 protein expression is highest after 48 hours of infection. Immunofluorescence microscopy has shown that during CMV infection these proteins accumulate in the juxtanuclear region of infected cells, with the Gbf1 protein also accumulating in the subplasmalemal region. Colocalization of Big1, Big2 and Gbf1 proteins with markers of the inner and outer part of the VAC showed that Big1, Big2 and Gbf1 proteins accumulate in the inner part of the VAC. Furthermore, Western blot, flow cytometry and immunofluorescence microscopy methods have shown that small inhibitors Brefeldin A (BFA), Golgicide A (GCA) and EXO2 impare establishment of MCMV infections and reduce viral protein expression. EXO2 was the most effective inhibitor although the effect of BFA showed the greatest statistical significance. Further research of the exact role of Big1, Big2 and Gbf1 proteins in the pathogenesis of CMV infection could contribute to its better understanding and thus better therapy.