Since the 1990s, a novel third approach to vaccination against a broad array of target antigens and diseases has been in development. This technology involves the direct administration of plasmid deoxyribonucleic acid (DNA) containing the gene encoding the immunogen against which an immune response is sought, leading to the in-situ production of the target immunogen(s) in the vaccine recipient. Such vaccines are referred to as “plasmid DNA vaccines” or simply “DNA vaccines” (with both terms used interchangeably throughout these Guidelines). The value and advantages of plasmid DNA products need to be assessed on a case-by-case basis; their utility will depend upon: (a) the nature of the organism being vaccinated against or the targeted disease; (b) the nature of the immunogen or activity of the gene insert; (c) the type of immune response required for effectiveness; and (d) the delivery system and route of administration. The development and application of DNA vaccines continues to progress. Since the WHO Guidelines for assuring the quality and nonclinical safety evaluation of DNA vaccines were adopted by the Expert Committee for Biological Standardization in 2005, many clinical trials of DNA vaccines have taken place and considerable experience in their manufacture and control has accrued. The current revision reflects this experience, especially in relation to data derived from nonclinical and clinical safety testing, which address many of the concerns expressed in previous versions of these Guidelines. The control of DNA vaccines should continue to be approached in a flexible manner to enable further modifications as more experience is gained in their production and use, and as other components or delivery systems are included. The intention of the current document is to provide a scientifically sound basis for the consistent manufacture and control of DNA vaccines for human use to ensure their continued safety and efficacy following licensure.