The effect of tunicamycin (Tm), a glycosylation inhibitor, on the epitopes expressed on Japanese encephalitis virus (JEV) glycoprotein E (gpE) in porcine kidney stable (PS) cells was studied. At Tm concentration of 2 micrograms/ml, the virus-infected cells showed markedly reduced or no reactivity with any of the monoclonal antibodies (MAbs) directed against JEV gpE except NHs-2 and also with polyclonal antibodies (PAbs) directed against JEV. With the increase in Tm concentration to 3 micrograms/ml, a complete loss of the conventionally detected reactivity of the MAbs except NHs-2 was recorded, while the Pabs showed no decrease in their reactivity. However, the MAb NHs-2 and PAbs lost their reactivity when the cells treated with 3 micrograms/ml Tm were stained for epitopes expressed on their surface indicating that glycosylation plays a role in this phenomenon. Tissue culture fluid (TCF) displayed a low virus content in the presence of 3 micrograms/ml Tm, indicating probably a down-regulation of virus maturation inside the cells. Since preM and NS-1 proteins possess besides gpE conserved N-glycosylation sites and play a role in the maturation of JEV, their expression in nascent, i.e. non-glycosylated form might be responsible for the observed low virus content of TCF. Thus, the glycosylation of JEV gpE seems essential for the acquisition of native conformation of its epitopes and their expression in cells.