The complete nucleotide sequence is presented of the 2 x 67 kbp BamHI-EcoRV portion of the BamHI 10 fragment of the pseudorabies virus (PRV) genome (strain Ka) containing sequences upstream of the previously reported protein kinase gene, and completing the sequence of this 4008 bp fragment. It is predicted to contain a gene designated RSp40, homologous to gene US1 of herpes simplex virus type 1 (HSV-1), with the potential to encode a protein of 364 amino acids. Analysis of PRV mRNA synthesized in the presence and absence of cycloheximide indicated that, in contrast to its HSV-1 homologue, the PRV gene RSp40 does not specify an immediate-early mRNA. Between the RSp40 gene and the protein kinase gene are two reiterated sequences: one containing 11 tandem copies of a 35 nucleotide sequence and the other containing nine tandem copies of a 10 nucleotide sequence. The BamHI 10 and the BamHI 12 fragments of PRV contain the junctions between the short unique (US) and short repeat (RS) regions of the PRV genome. The nucleotide sequence of that portion of the BamHI 12 fragment containing US sequences was determined so that, by comparison with the nucleotide sequence of the BamHI 10 fragment, the junction between the US and RS regions could be defined. In BamHI 10 this was found to be at a point between the two reiterated sequences (which are in the RS region) and the protein kinase gene (which is in the US region). The organization of this region of the PRV genome is compared to that of other alphaherpesviruses.