An important limitation of El-deleted recombinant adenoviruses in gene therapy is the immune response that they engender and that rapidly destroys transduced cells. Transduced cells of the outer retina appear to be an exception. To determine whether differences in immune sequestration of the outer retina contribute to the increased stability of transgene expression in this tissue, we examined the systemic humoral response to an El-deleted adenovirus injected into the subretinal space. Subretinal injection of Ad.CMVlacZ in mature immunocompetent mice resulted in minimal circulating antibody production. In contrast, subcutaneous administration of equivalent doses of Ad.CMVlacZ resulted in high-titer antibody production against adenoviral proteins. Circulating antibodies in systemically immunized animals had minimal effect on retinal transgene expression patterns measured 2 weeks after subretinal injection of Ad.CMVlacZ. Histologic examination showed minimal retinal toxicity attributable to subretinal adenovirus in naive or immunized mice, although 3/18 (14%) of eyes from the latter set contained a localized granulomatous infiltrate at the ocular injection site. The data demonstrate that the subretinal space is an immune-privileged site regarding humoral immunity. Further, short-term transduction efficiency is not affected by the presence of antiadenoviral antibodies. The retina may be a favorable environment for replication-defective virus-mediated genetic therapy.