A late gene of the Hz-1 insect virus (Hz-1V) encoding a predicted polypeptide of 34 kilodaltons (kDa) was isolated from a cDNA library and mapped to the HindIII-T region (50.3 to 52.4 map units) of the viral genome. The p34 gene was characterized by DNA sequence, Northern blot, and primer extension analyses. The 765-bp open reading frame (ORF) is transcribed in the clockwise direction as a 1.2-kb RNA. Primer extension analysis detected two late transcription initiation sites at -16 and -17 nt relative to the start of the p34 ORF. Transcription initiation was observed between 4 and 18 hr postinfection (hr p.i.) with maximum expression at 12 hr p.i. No nucleotide sequence homology was detected between the regulatory region of the p34 gene and the baculovirus conserved late promoter motif NTAAG. This observation was substantiated by results obtained from an investigation of Hz-1V late gene expression using a transient expression assay system which suggested that Hz-1V late gene promoters do not resemble the baculovirus late promoter motif. This is the first molecular analysis of Hz-1V late gene expression and offers a basis by which to compare Hz-1V to other insect viruses.