Poxviruses carry the enzyme, nucleoside triphosphate phosphohydrolase I (NPH I), required for early viral transcription in the cytoplasm of infected cells. The gene (nph I) encoding this enzyme from Choristoneura fumiferana entomopoxvirus (CfEPV) has been located in the viral genome, cloned and characterized. It has an open reading frame of 1941 nucleotides, potentially encoding a protein with a predicted molecular mass of 76.04 kDa and a pI of 8.83. It has a TAAATG motif where the trinucleotide ATG represents the translational start signal an AT-rich (88%) sequence and an early transcription termination signal (TTTTTAT) upstream of the ATG codon. Northern blot analysis of mRNA from infected larvae showed that a single 4.0 kb transcript which appeared late at day 20 post infection (p.i.) and its transcription continued till day 37 p.i.. Primer extension experiments suggested that the main transcripts started at 15 bases upstream of AUG codon. NPH I homologues have been found in the genomes of other entomopoxviruses and vertebrate poxviruses. Alignment of their amino acid sequences suggested three conserved domains, two of which are considered as ATP binding domains. The most similar homologue is from the closely related entomopoxvirus. Choristoneura biennis EPV (CbEPV) where 98.2% of nucleotide and 97.2% of amino acid identities are observed, respectively. A single nucleotide difference in CfEPV nph I was sufficient to distinguish it from CbEPV by PCR amplification and digestion with a restriction enzyme.