Aim of the study: Cytomegaloviruses (CMV) initiate the stepwise establishment of the cytoplasmic virion assembly compartment (cVAC) from the earliest stages of infection by the expansion of the early endosome (EE)/endosomal recycling compartment (ERC) interface and relocation of the Golgi complex. This membranous organelle reorganization also affects phosphatidylinositol 3-phosphate (PI(3)P)-containing membrane domains. Since PI(3)P is the primary regulator of the EE pathway, this study aimed to examine the role of PI(3)P in the physiology of the EE system and the contribution of PI(3)P-dependent EE domains in cVAC biogenesis and progression of the entire CMV life cycle. Material and methods: Functional analysis of PI(3)P was based on its pharmacological depletion using VPS34-IN1, an inhibitor of the major PI(3)P producer on EE (Vps34), and on inhibition of PI(3)P-dependent functions by overexpression of recombinant PI(3)P-binding domains. Under such conditions, we monitored changes in the physiology of the EE pathway and the process of cVAC development through the expression of its markers by confocal microscopy. With Western blot analysis, viral DNA synthesis, and CMV growth analysis, we examined CMV replication cycle progression and the ability to efficiently assemble and release newly synthesized virions. Results: PI(3)P depletion in uninfected and infected cells led to the rapid reorganization of the Rab11a-dependent endosomal pathway, as evidenced by the spread and vacuolization of Rab11a endosomes in the perinuclear region and the disappearance of the ERC. The PI(3)P depletion also induced the separation of transferrin+ and Rab11a+ endosomal vesicles, which indicates the PI(3)P role in recycling cargo sorting and EE to ERC transport. Although PI(3)P+ and Vps34+ endosomes build a substantial part of cVAC, the PI(3)P inhibition did not prevent the establishment of infection, progression through the early phase of infection, and membrane reorganization related to the cVAC development. However, PI(3)P depletion resulted in viral growth inhibition, and a reduced number of virions released was associated with the inhibition of viral DNA replication and the expression of late viral proteins. Conclusion: PI(3)P-dependent functions, including the Rab11-dependent EE-to-ERC route, are dispensable for the establishment of cVAC. Nevertheless, drastically reduced viral growth under PI(3)P depletion and significantly inhibited late infection events suggest that PI(3)P is essential for the entry into the late phase of infection and the final formation of new virions.