Tunicamycin added in concentrations of 0.5-1 microgram/ml into the maintenance medium with tick-borne encephalitis virus (TBE) propagated in PEK cell culture inhibited incorporation of labeled carbohydrates into glycoprotein V3 as well as into virus-specific proteins NV41/2, NV3, p22, p18, p13, and p12 and to a lower degree into proteins NV5 and NV4 irrespective of the time of its addition (immediately or 17 hours after virus adsorption on the cells). In the presence of tunicamycin, incorporation of radioactive amino acids into the intracellular virus-specific proteins was inhibited, and proteins with altered electrophoretic mobility appeared. Tunicamycin in doses of 0.5-1 microgram/ml added to the infected cells immediately after virus adsorption reduced the production of extracellular virus by 2 lg PFU/ml and of intracellular virus by 1 lg PFU/ml, but exerted no inhibitory effect on virus production when added 17 hours after infection. The results indicate that protein V3 and probably all other intracellular virus-specific proteins of TBE virus exist in PEK cells in N-glycosylated form, tunicamycin selectively inhibits the stage of glycosylation of virus-specific proteins, and glycosylation of proteins affects the infectious properties and virus release from the cell.