We mutagenized and characterized a 41-kilobase-pair subgenomic cloned fragment from the unique long (UL) region of pseudorabies virus (PRV). Forty mutant clones, each carrying a single inserted oligonucleotide, were used for cotransfection with overlapping cloned viral DNA fragments. More than half of these transfections yielded viable virus mutants. Short viral DNA fragments, flanking the oligonucleotide insertions, were cloned and used as probes on Northern blots with RNA isolated from cells infected with wild-type PRV. In this way we were able to construct a partial transcriptional map of this region of the PRV genome. In addition, we used these probes in cross-hybridization studies with cloned genomic fragments from the prototype alphaherpesvirus herpes simplex virus type 1 (HSV-1). This allowed us to define homology between the corresponding regions of these viruses. Most viable PRV mutants were assayed for virulence in mice. Mutagenesis of the identified homologs of HSV-1 genes UL39 (encoding the large subunit of ribonucleotide reductase), UL40 (small subunit of ribonucleotide reductase), UL42 (DNA polymerase accessory factor), UL23 (thymidine kinase), UL21 (a capsid-associated protein), and UL12 (alkaline nuclease) completely abrogated or strongly reduced virulence.