The Epstein-Barr virus (EBV) infects humans and the genome of this infectious agent has been detected in several tumour types, ranging from lymphomas to carcinomas. The analysis of the functions of the numerous viral proteins encoded by EBV has been impeded by the large size of the viral genome, which renders the construction of viral mutants difficult. To overcome these limitations, several genetic systems have been developed that allow the modification of the viral genome. Two different approaches, depending on the host cell type in which the viral mutants are generated, have been used in the past. Traditionally, mutants were constructed in EBV infected eukaryotic cells, but more recently, approaches that make use of a recombinant EBV cloned in Escherichia coli have been proposed. The phenotype associated with the inactivation or modification of nearly 20 of the 100 EBV viral genes has been reported in the literature. In most of the reported cases, the EBV latent genes that mediate the ability of EBV to immortalize infected cells were the targets of the genetic analysis, but some virus mutants in which genes involved in DNA lytic replication or infection were disrupted have also been reported. The ability to modify the viral genome also opens the way to the construction of viral strains with medical relevance. A cell line infected by a virus that lacks the EBV packaging sequences can be used as a helper cell line for the encapsidation of EBV based viral vectors. This cell line will allow the evaluation of EBV as a gene transfer system with applications in gene therapy. Finally, genetically modified non-pathogenic strains will provide a basis for the design of an attenuated EBV live vaccine.