The complete genome of Marek's disease virus serotype 1 (MDV-1) strain 584Ap80C was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the U(S)2 locus of the MDV-1 genome by homologous recombination. Viral DNA containing the BAC vector was used to transform Escherichia coli strain DH10B, and several colonies harboring the complete MDV-1 genome as an F plasmid (MDV-1 BACs) were identified. DNA from various MDV-1 BACs was transfected into chicken embryo fibroblasts, and from 3 days after transfection, infectious MDV-1 was obtained. Growth of MDV-1 recovered from BACs was indistinguishable from that of the parental virus, as assessed by plaque formation and determination of growth curves. In one of the MDV-1 BAC clones, sequences encoding glycoprotein B (gB) were deleted by one-step mutagenesis using a linear DNA fragment amplified by PCR. Mutant MDV-1 recovered after transfection of BAC DNA that harbored a 2.0-kbp deletion of the 2.6-kbp gB gene were able to grow and induce MDV-1-specific plaques only on cells providing MDV-1 gB in trans. The gB-negative virus reported here represents the first MDV-1 mutant with a deletion of an essential gene and demonstrates the power and usefulness of BACs to analyze genes and gene products in slowly growing and strictly cell-associated herpesviruses.