Diphtheria toxin (DT) is an extracellular protein of Corynebacterium diphtheriae that inhibits protein synthesis and kills susceptible cells. The gene that encodes DT (tox) is present in some corynephages, and DT is only produced by C. diphtheriae isolates that harbor tox+ phages. The diphtheria toxin repressor (DtxR) is a global regulatory protein that uses Fe2+ as co-repressor. Holo-DtxR represses production of DT, corynebacterial siderophore, heme oxygenase, and several other proteins. Diagnostic tests for toxinogenicity of C. diphtheriae are based either on immunoassays or on bioassays for DT. Molecular analysis of tox and dtxR genes in recent clinical isolates of C. diphtheriae revealed several tox alleles that encode identical DT proteins and multiple dtxR alleles that encode five variants of DtxR protein. Therefore, recent clinical isolates of C. diphtheriae produce a single antigenic type of DT, and diphtheria toxoid continues to be an effective vaccine for immunization against diphtheria.