To optimize the in vitro production of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) as a potential microbial pest control agent, the pathogenicity of occlusion bodies (OBs) produced in two cell lines at three incubation temperatures was determined by bioassay. A plaque-purified isolate of CfMNPV was amplified in permissive C. fumiferana cell lines, FPMI-CF-203 and FPMI-CF-2C1, and incubated at 22, 24, and 28° C. Occlusion bodies propagated in FPMI-CF-203 cells at 28° C were significantly larger (17.5 μm³) and more pathogenic (LD₅₀ = 27; LD₉₅ = 185, where LD₅₀ and LD₉₅ are doses required to kill 50 and 95% of the test larvae, respectively) than those produced in either of the cell lines at any of the incubation temperatures tested. Increased temperatures yielded larger OBs from both cell lines. The pathogenicity of OBs propagated in the FPMI-CF-203 cell line increased with incubation temperature, whereas that of OBs produced in FPMI-CF-2C1 cells decreased. Comparison of the pathogenicity of OBs, whether naturally occurring or genetically modified, should be standardized by cell line and incubation temperature used for propagation. Production efficiency decreased with increasing incubation temperature for each cell line. Lower incubation temperatures used for propagation, and standardization of the titer of viral inoculum, should be further investigated to determine the economic feasibility of the in vitro production of CfMNPV as a microbial pest control agent.