In this report, the open reading frame 21 (Bm21) of Bombyx mori nucleopolyhedrovirus (BmNPV), one of the unique genes of group I NPVs, was characterized. Bm21 is predicted to encode a protein of 55.8 kDa and was found to contain imperfectly conserved leucine-rich repeats. 3' Rapid amplification of cDNA ends (3'RACE) showed that the transcript of Bm21 was first detected from 6 h post-infection and that it also encompassed the complete Bm20. 5'RACE revealed three transcription initiation sites, one of which mapped to the baculovirus early transcription motifs CGTGC and CAGT. Transient-expression and superinfection assays indicated that BM21 localized in the nucleus of infected BmN cells. To study the function of BM21, a Bm21-null virus was constructed using bacmid technology. Viral one-step growth curve analyses showed that the Bm21-null virus had similar budded virus production kinetics to those of the parental virus. Bioassay analyses showed that the median lethal concentration (LC(50)) of the Bm21-null virus was similar to that of the control virus; however, the median survival time (ST(50)) of the knockout virus was significantly longer than the control virus. These results indicate that BM21 is not essential for virus replication in vitro, but that deletion of the gene delays the killing of the infected larvae.