A trans-acting gene required for late viral gene expression in transient expression assays was identified in the genome of Autographa californica nuclear polyhedrosis virus. A genomic library of A. californica nuclear polyhedrosis virus DNA lacking a clone spanning the region from 43 to 48 map units (mu) was unable to activate gene expression from a reporter plasmid when the reporter gene was driven by a baculovirus late or very late promoter in transient expression assays. The genomic region responsible for activating reporter gene expression was further mapped to 43.4 to 45.2 mu of the viral genome. The nucleotide sequence of this region was determined and shown to contain several small open reading frames (ORFs) and one major ORF, named lef-3, for late expression factor 3. The lef-3 ORF was predicted to encode a polypeptide of 385 amino acids and with a molecular mass of 44,529 daltons. No homolog to the lef-3 ORF was found in existing data bases. Further analysis showed that only the lef-3 ORF in the region from 43.4 to 45.2 mu was necessary for late gene activation in the assays used. The temporal regulation of lef-3 transcription was studied by Northern (RNA) blot hybridization; lef-3 was found to be an early gene that was transcribed primarily as a 2.0-kb mRNA. Primer extension analysis and S1 nuclease protection assays revealed that lef-3 transcription initiated about 280 bp upstream of the first ATG codon and terminated near a polyadenylation signal, 130 bp downstream of the last codon of the lef-3 ORF.