Orf68 (ac68) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is identified to be an early gene, but its transcription start site remains unknown. The coding sequence of ac68 overlaps 280-bp leading sequence and 159-bp coding sequence of lef3 (ac67). In this study, the transcription start site of ac68 was determined by 5' RACE analysis to be 18 nucleotides upstream from the start codon. In order to investigate the effect of ac68 deletion on virus propagation, we generated a bacmid with an ac68 knockout by deleting 360-bp inside the ac68 gene, which also deleted 220-bp leading sequence of lef3. Production of infectious budded virus and formation of nucleocapsids and occlusion bodies exhibited wild-type patterns of virus propagation in Sf-9 cells infected with the mutant bacmid. The result demonstrated that ac68 was not an essential gene for viral propagation which was confirmed by further deletion of ac68, and disruption of the lef3 leading sequence did not affect viral propagation. Ac68 was the second auxiliary gene discovered besides Ac133 (alk-exo) among the 30 core genes of AcMNPV.