Sequence analysis of a 6.4 kb DNA region from the Spodoptera littoralis multinucleocapsid nucleopolyhedrovirus (SpliNPV) revealed a large open reading frame (ORF) encoding a predicted polypeptide of 998 amino acid (aa) residues with a molecular mass of 114.93 kDa, located between 47.2-52.3 m.u. on the SpliNPV genome. Comparative sequence analyses demonstrated that the ORF encodes a DNA polymerase gene (dnapol) that contains conserved exonuclease domains and DNA polymerase motifs found in many prokaryotic, eukaryotic, and viral replicative DNA polymerases. A second ORF, ORF138, located between the lef-3 and dnapol, encodes a 138 aa polypetide that is homologous to ORF66 of the Autographa californica MNPV (AcMNPV). SpliNPV DNA polymerase shares an overall aa sequence identity of 39% with that of AcMNPV. A 3.0 kb SpliNPV dnapol-specific transcript was detected initially at 2 hpi and became abundant 48 hpi by Northern blot analysis. The transcription initiation site was mapped to an NPV early promoter element, ACGT. 3' RACE demonstrated that the SpliNPV dnapol transcript terminated at the polyadenylation signal AATAAA. Sequence analysis suggested that the SpliNPV dnapol and the dnapol of the NPV of S. litura (SpltNPV) are closely related.