The Spodoptera littoralis multicapsid nucleopolyhedrovirus (SpliMNPV) shows only a distant genetic relationship to other NPVs. In this report we describe the gene organization of an 8-kbp region of the SpliMNPV which contains the polyhedrin gene. The polyhedrin transcription initiation sites were mapped and the sequence and gene organization of an 8-kbp region of SpliMNPV were determined. The sequences downstream of the polyhedrin gene showed colinearity with the gene organization of other NPVs. An anticlockwise 1035-bp open reading frame (ORF), capable of encoding a proline-rich polypeptide, was found at the 3' end of the polyhedrin gene. followed by an 837-bp ORF encoding a putative protein kinase (PK), with an orientation similar to that of the polyhedrin gene. Sequences upstream of the polyhedrin gene were found to be unique to SpliMNPV and contained two regions consisting of highly repetitive sequences. One region, 980 bp in length and termed sequence repeat region 1 (SR1), contained a variety of short direct repeats, SR1 was found to act as a transcriptional enhancer in a transient expression assay. Additional regions containing different repetitive sequences were identified within the proline-rich ORF1035 and in sequences located downstream of the pk gene.