The replication of vesicular stomatitis virus (VSV) is inhibited by tunicamycin (TM), an antibiotic that blocks the formation of N-acetylglucosaminelipid intermediates. We had shown previously that the viral glycoprotein (G) synthesized in cells treated with TM is not glycosylated and is not found on the outer surface of the cell plasma membrane. In this report, we shown that cells exposed to TM produce a low yield of infectious particles. The yield is increased when the temperature during infection is lowered from 37 to 30 degrees C. At 30 degrees C in the presence of TM, both wild-type VSV and the temperature-sensitive mutant ts045 produce particles that do not bind to concanavalin A Sepharose and contain only the nonglycosylated form of G. These particles have a specific infectivity (pfu/cpm) comparable to that of VSV containing glycosylated G.