Subretinal delivery of recombinant adeno-associated virus (rAAV) results in a systemic humoral response in the adult immunocompetent mouse. We characterized this response and determined whether it is possible to readminister rAAV to the subretinal space despite the presence of antibodies to the vector. A systemic humoral response to rAAV capsid proteins was induced by either unilateral subretinal injection or by intradermal administration of 1 x 10(9) infectious units of rAAV carrying the cDNA encoding green fluorescent protein (GFP), rAAV-GFP. Experiments were performed in cohorts of adult C57BL/6 mice. Assessment of systemic humoral response to viral capsid protein was performed through enzyme-linked immunoabsorbent assay (ELISA) and infection inhibition studies of serum samples 3 weeks after virus delivery. The rAAV-GFP virus was readministered by subretinal injection. GFP expression after subretinal administration was evaluated ophthalmoscopically throughout the course of the experiment and histologically at the termination of the experiment. We observed significant systemic humoral responses to viral capsid protein after subretinal delivery of rAAV. Intradermal injection resulted in a larger humoral response (with a higher percentage of neutralizing antibodies) than subretinal injection. Additional transduction events were observed after readministration of rAAV despite the presence of strong humoral response to the vector.