The ORF38 of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), or AcORF38, contains a conserved motif of Nudix (nucleotide diphosphate X) superfamily. It has the highest homology with ADP-ribose pyrophosphatase (ADPRase), a subfamily of Nudix pyrophosphatase. In the current study, recombinant AcORF38 protein was prepared and shown to have ADPRase activity, with a Km of 204 microM, and K(cat) of 6.96 s(-1) at pH 8.0 and 5 mM MgCl2. The transcription of AcORF38 was detected 2 h postinfection, and lasted until the late stage. An orf38 gene-deleted mutant virus, vAcGFP-Delta38, was constructed. Although it produced progeny virus, the yield of extracellular virus was less than 1% of the wild-type virus. The activity of viral very late gene promoter was also greatly reduced in vAcGFP-Delta38-infected cells as indicated by the expression of green fluorescence protein gene driven by polyhedrin promoter. The mutant phenotype was rescued by co-transfection with an AcORF38-expressing plasmid. These results suggest that AcORF38 plays an important role in virus replication, although the detail is to be elucidated.