Herpes simplex virus (HSV) is a neurotropic DNA virus. The viral genome is large (152 kb), and many genes are dispensable for viral function, allowing insertion of multiple or large transgene expression cassettes. The virus life cycle includes a latent phase, during which the viral genome remains as a stable episomal element within neuronal nuclei for the lifetime of the host, without disturbing normal function. We have exploited these features of HSV to construct a series of nonpathogenic gene therapy vectors that efficiently deliver therapeutic and experimental transgenes to neural and non-neural tissue. Importantly, transgene expression may be sustained long term; reporter gene expression has been demonstrated for over a year in the nervous system. This article discusses the generation of replication-defective HSV vectors and reviews recent studies investigating their use in several animal models of human disease. We have demonstrated correction or prevention of a number of important neurological phenotypes, including neurodegeneration, chronic pain, peripheral neuropathy, and malignancy. In addition, HSV-mediated transduction of non-neurological tissues allows their use as depot sites for synthesis of circulating and locally acting secreted proteins. New applications for this vector system include the genetic modification of stem cell populations; this may become an important means to direct cellular differentiation or deliver therapeutic genes systemically. Replication-defective HSV vectors are an effective and flexible vehicle for the delivery of transgenes to numerous tissues, with multiple applications.