The aim of this study was to confirm or refute the difference between efficacy of long-term specific immunotherapy (SIT) with standardized allergen vaccine consisting of six grass pollens (oat grass, orchard grass, fescue, rye grass, timothy grass, and rye) administered either by sublingual or by supralingual route. To investigate clinical and immunologic changes, 51 patients of a previous 1-year double-blind, placebo-controlled, randomized study were enrolled in an open randomized study that continued over the next 3 years. Sublingual or supralingual immunotherapy (SLIT) was performed in the same way, keeping the drops under or on the tongue, respectively, for 1-2 minutes before swallowing them. Data about symptoms scores and rescue medication intake during grass pollen seasons, as well as skin-prick test results, levels of specific IgG, and IgE antibodies were collected after each pollen season. It was clearly shown that both routes of administration were effective, leading to a significant decrease of clinical symptoms of grass pollen allergy after SIT lasting 3-4 years. No statistically significant difference between sublingually and supralingually treated patients was observed at the end of the study. Adverse effects were limited to a small number of generally mild local and/or systemic reactions with no significant difference between both administration ways of SIT. The significant therapeutic effect of both SLIT and supralingual immunotherapy lasting 3-4 years was clearly achieved. Despite no significant difference between efficacy of both administration ways of SIT, the onset of sublingual SIT effect seems to be slightly faster than that of supralingual SIT.