Corneal transplantation is the most frequent and successful form of tissue transplantation in adults (<10% rejection). In young children, any corneal opacity should be corrected as early as possible to prevent lifelong visual impairment. However, the corneal graft rejection rate is dramatically increased in infants younger than 12 months of age (up to 85% rejection), and immunosuppressive therapy is particularly challenging in this age group. Regulatory T cells (Tregs) are a well-characterized T cell subpopulation with the potential to prevent autoimmune disorders or transplant rejection. Antigen-specific Tregs were shown to inhibit graft rejection in adult stem cell transplantation. Less is known about the role of naïve Tregs.The purpose of the present study was to elucidate the relevance of naïve Tregs in juvenile corneal transplantation in a baby rat keratoplasty model that reproduces the accelerated rejection in young patients. Counts and inhibitory potential of Tregs were studied in spleens of 3- and 10-week-old rats. Unprimed Tregs (CD4+CD25+) were isolated from the spleens of 10-week-old Lewis rats and systemically or subconjunctivally administered in vivo in allogenic keratoplasty in 3- and 10-week-old Lewis recipient rats. In subconjunctival tissue, transcription was analyzed for induction of transforming-growth-factor beta (TGF-β). In 3-week-old rats, CD4 T cell counts, but not FoxP3 T cell counts were lower than in 10-week-old rats. The Tregs of both age groups had the potential to inhibit T cell activation in vitro. No significant delay in rejection was observed when Tregs were applied systemically before keratoplasty. However, subconjunctival application of Tregs abrogated rejection in 66.7% and 33.3% of the 3- and 10-week-old recipients, respectively. Analysis of the conjunctival tissue revealed a transplantation-induced increase in TGF-β transcription in the 3-week-old rats. Our data suggest that local application of unprimed regulatory T cells may be a therapeutic strategy for preventing corneal graft rejection in young recipients.