Purpose: We tested whether antigen administration via the anterior chamber (a.c.) was equivalent to intravenous (i.v.) or mucosal administration of antigen. Methods: Ovalbumin (OVA)-specific CD8+ T cells (OT-I) were enumerated in lymphoid tissues of C57B1/6 (B6) mice via adoptive transfer after the same amount of antigen was administered via a.c., i.v., or mucosal routes. Lytic activity was measured in B6 and γδT cell-deficient B6 mice given OVA via a.c., i.v, or mucosal routes after injection with OVA in adjuvant. Results: OVA a.c. induced a pattern of T-cell proliferation distinct from i.v. or mucosal administration. A.c. and i.v., but not mucosal, OVA induced cytolytic T lymphocyte (CTL) tolerance. The inhibition of CTL responses was significantly greater in mice given OVA a.c. rather than i.v. γδT cells contributed to a.c.-, but not i.v.-, induced CTL tolerance. Conclusions: A.c. administration of antigen is not de-facto i.v. or mucosal administration of antigen. Copyright © Taylor & Francis Inc.