The 150-kbp genome of the alphaherpesvirus equine herpesvirus 1 (EHV-1) strain HVS25A was cloned as a bacterial artificial chromosome (EHV-1 BAC), with mini F plasmid sequences inserted between genes 62 and 63. Transfection of EHV-1 BAC DNA purified from E. coli gave rise to progeny virus that had a similar growth rate and yield in mammalian cell culture to those of parental wild-type EHV-1. Using in vitro mutagenesis with a Mu transposon, a large library of EHV-1 BAC mutants was generated, and sequence analysis indicated that insertions were dispersed randomly across the EHV-1 genome. Following transfections of a pilot sample of mutant EHV-1 BAC DNAs into mammalian cells, no CPE was observable by light microscopy for mutants carrying insertions in genes for the major capsid protein, large tegument protein, glycoprotein K, catalytic subunit of DNA polymerase, or single-stranded DNA-binding protein. Mutants that were able to produce CPE similar to wild-type EHV-1 included those with interruptions in ORFs of several tegument proteins. Analysis of several glycoprotein gene mutants indicated that those carrying insertions near the start of genes encoding glycoproteins E and I were viable, but showed markedly diminished plaque areas. These results were supported by confocal microscopy of transfected or infected cultures. Electron microscopy of cells infected with a gE mutant revealed accumulations of particles within cytoplasmic vesicles, consistent with a partial obstruction of maturation. The transposon library is a resource for comprehensive functional analysis of the HVS25A genome, with multiple mutants available in any of the predicted genes of EHV-1.