The baculovirus GP64 envelope fusion protein (GP64 EFP) is a class I integral membrane protein that enters the secretory pathway and is oligomerized and extensively processed during transport to the plasma membrane. The kinetics of GP64 EFP biosynthesis, oligomerization, and processing in Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV)-infected Lymantria dispar cells were examined by pulse label, pulse-chase, and immunoprecipitation experiments. Relative rates of GP64 EFP synthesis in OpMNPV-infected L. dispar cells were examined at various times throughout the infection cycle. Using pulse labeling and immunoprecipitation, GP64 EFP synthesis was detected within 2 hr p.i., and the maximal rate of synthesis was observed in the period of 24-26 hr p.i., a time coincident with the onset of high level production of budded virus in OpMNPV-infected L. dispar cells. To determine the oligomeric structure of GP64 EFP, a soluble form of OpMNPV GP64 EFP was produced and examined by a combination of gel filtration chromatography, nonreducing SDS-PAGE, and mass spectrometry. Oligomeric GP64 EFP was identified as a trimeric molecule, that migrates as two discrete bands on nonreducing SDS-PAGE. Pulse-chase studies, performed at both early (12 hr p.i.) and late (36 hr p.i.) stages of the infection cycle, showed that GP64 EFP oligomerization is complete within 15 min after synthesis. Efficiency of oligomerization however was relatively low, with less than 33% of the synthesized GP64 EFP converted to trimers. The majority of monomeric GP64 EFP remaining in the cell appeared to be degraded within 30 to 45 min after synthesis. Analysis of the kinetics of carbohydrate processing at early (12 hr p.i.) and late (36 hr p.i.) times postinfection showed that for both early and late phases of infection, carbohydrate was rapidly added, and processing began between 10 and 20 min after GP64 EFP synthesis. Although carbohydrate processing was completed within approximately 90 min after synthesis during the early phase, the same process required approximately 150 min during the late phase. Thus, carbohydrate processing appeared to become less efficient as infection progressed. These studies thus show that GP64 EFP undergoes a rapid but inefficient oligomerization step that results in a homotrimeric structure for GP64 EFP. While carbohydrate addition is rapid, carbohydrate processing requires prolonged periods of time (with half-times of 45 to 75 min) and appears to become less efficient during the late phase of the infection.