Influenza viruses bud from the plasma membrane of virus-infected cells. Although budding is a critical step in virus replication, little is known about the requirements of the budding process. In this report, we have investigated the role of ATP in influenza virus budding by treating influenza virus infected Madin-Darby canine kidney (MDCK) cells with a number of metabolic inhibitors. When WSN virus-infected MDCK cells were exposed to antimycin A, carbonyl cyanide m-chlorophenylhydrazone, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, or oligomycin for a short time (15 min or 1 h) late in the infectious cycle, the rate of virus budding decreased. This inhibitory effect was reversible upon removal of the inhibitors. The role of ATP hydrolysis was analyzed by treating lysophosphatidylcholine (LPC)-permeabilized live filter-grown virus-infected MDCK cells with nonpermeable ATP analogues from the basal side and assaying virus budding from the apical side. In LPC-permeabilized cells, membrane-impermeable ATP analogues such as adenosine 5'-O-(3-thiotriphosphate) or 5'-adenylylimidodiphosphate caused reduction of virus budding which could be partially restored by adding excess ATP. These data demonstrated that ATP hydrolysis and not just ATP binding was required for virus budding. However, inhibitors of ion channel (ATPases) and protein ubiquitinylation, which also required the ATP as energy source, did not affect influenza virus budding, suggesting that neither ion channel nor protein ubiquitinylation activity was involved in influenza virus budding. On the other hand, treatment with dimethyl sulfoxide (DMSO), which decreases membrane viscosity, reduced the rate of virus budding, demonstrating that the physical state of membrane viscosity and membrane fluidity had an important effect on virus budding. Data presented in the report indicate that influenza virus budding is an active ATP-dependent process and suggest that reduced virus budding by ATP depletion and DMSO treatment may be partly due to decreased membrane viscosity.