To evaluate the role of cell-mediated immunity in the stability of ocular adenovirus-mediated transgene expression. Adenovirus (4 x 10 (6) pfu) containing lacZ (Ad. CMVlacZ) was injected intravitreally or subretinally into one or both eyes of immunocompetent (+/+) and immunocompromised (nu/nu) CD-1 mice. Control eyes received injections of saline. Additional+/+ mice received subretinal injections of Ad. CMVlacZ with coadministration of 200 microg of human immunoglobulin (Ig) G or CTLA4Ig by intraperitoneal, intravitreal, or subretinal injection. The mice were killed at various times after injection, and their eyes were examined histologically and immunohistochemically. LacZ expression was extended from 1 week to more than 5 weeks in the corneal endothelium, iris, and trabecular meshwork of nu/nu mice compared with time of expression in+/+ mice when adenovirus was administered intravitreally. In contrast, subretinal injection resulted in only a minimal increase in transgene stability in nu/nu mice compared with that in+/+ mice. Neither systemic nor intraocular administration of IgG or CTLA4Ig affected the stability of lacZ expression in the retina or retinal pigment epithelium after subretinal injection in+/+ mice. Immunoglobulin G and CTLA4Ig enhanced the stability of transgene expression in the trabecular meshwork. A T-cell-mediated immune response appears to play a role in the loss of adenovirus-mediated lacZ expression after intravitreal but not after subretinal injection. This result implies that the subretinal space is an immune-privileged site and a favorable site for gene transfer.