Purpose. To determine whether differences in immune sequestration of the outer retina may explain the increased stability of transgene expression in this tissue. Methods. The systemic humoral response to injection of 5 × 108 pfu of the E1-deleted virus Ad.CMVlacZ (provided by I. Wilson) was measured by Western blot analysis of serum samples. Comparisons were made between the responses after intradermal injection and after injection into the subretinal space. After necropsy, histochemical assays were performed for analysis of β-galactosidase activity. Inflammatory response was assessed by examination of hematoxylin and eosin-stained sections. Results. Subretinal injection of Ad.CMVlacZ in immunocompetent mice resulted in no significant antibody production whereas systemic administration of Ad.CMVlacZ induced the production of circulating antibodies. Circulating antibodies had no 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 contained a localized macrophage collection at the ocular injection site. Conclusions. The decreased immune surveillance in the outer retina suggests that this tissue may be better suited than others for replication-defective virus-mediated somatic gene therapy.