PURPOSE: To determine the afferent pathways linking the anterior chamber (AC) of the eye to the secondary lymphoid organs. METHODS: Single intracameral, subconjunctival, or intravenous injections and topical application on the conjunctiva of 3 muL (30 mug) of cascade-blue-labeled Dextran (CB-Dx) were performed in Lewis rats. In addition, bilateral intracameral injections (CB-Dx into the right AC and FITC-Dx into the left AC) or a combination of intracameral (CB-Dx) and intravenous (FITC-Dx) injections were performed. Distribution of antigen-positive cells and free antigen in frozen sections of lymphoid organs from animals killed at 24 hours after these various types of injection was analyzed by fluorescence and confocal microscopy. RESULTS: After intracameral and subconjunctival injections, antigen reaches the ipsilateral lymph node of the head and neck predominantly via the conjunctival lymphatics. Intraocular antigen entering the venous circulation reaches the spleen and mesenteric lymph nodes, but also a small proportion enters the lymph nodes of the head and neck. After bilateral intracameral injections, individual cells bearing both fluorescent antigens were identified in lymphoid organs draining the eye. Similarly, double antigen-positive marginal zone macrophages were observed after simultaneous intracameral injection of CB-Dx and intravenous injection of FITC-Dx. CONCLUSIONS: These data demonstrate that in the first 24 hours after injection of antigen into the anterior chamber of the eye, antigen reaches the lymphoid organs mainly in a soluble form via both the blood and lymph.