We describe the isolation and characterization of infectious pseudorabies virus (PrV) mutants expressing functional beta-galactosidase. To obtain high level expression of the enzyme, sequences of the bacterial beta-galactosidase gene starting with codon 8 were inserted in frame behind the promoter and the first seven codons of the nonessential PrV glycoprotein gX-gene. Cotransfection of this construct with viral DNA yielded PrV mutants that could be easily identified after plaque staining with a chromogenic substrate. These mutants carry the gX-beta galactosidase fusion gene inserted into the authentic gX-gene leading to loss of gX-expression. The gX-beta galactosidase fusion gene could be excised as an expression cassette and placed into other non-essential PrV genomic regions, such as the thymidine kinase gene and the glycoprotein gI-gene, resulting in inactivation of the target genes. The fusion gene remains stably integrated in the viral genome at all three locations tested. It therefore appears ideal as an insertional and easily identifiable marker and greatly facilitates isolation and purification of PrV mutants.