To date, there have been no randomized, double-blind studies showing the effectiveness of sublingual immunotherapy with multiple allergens. The purpose of this study was to examine whether the efficacy of sublingual immunotherapy (SLIT) with standardized timothy extract was reduced by combination with other allergen extracts. A single-center, randomized, double-blind, placebo-controlled trial with SLIT was conducted. After an observational grass season, SLIT was administered for 10 months to 54 patients randomized to 1 of 3 treatment arms: placebo, timothy extract (19 microg Phl p 5 daily) as monotherapy, or the same dose of timothy extract plus 9 additional pollen extracts. Symptom and medication scores were collected and titrated nasal challenges, titrated skin prick tests, specific IgE, IgG4 and cytokines release by timothy-stimulated lymphocyte proliferation were performed. Perhaps because of a very low grass pollen season in 2008, there were no significant differences in medication or symptom scores in either treatment group compared with placebo. Compared with placebo, in the timothy monotherapy group, thresholds for titrated nasal challenge and skin prick tests (P = .03 and P = .001, respectively), and serum-specific IgG4 levels (P = .005) significantly increased, and IFN- gamma levels decreased (P = .02), whereas in the multiallergen group, there was significant improvement only in the titrated skin prick tests (P = .04) which was less than in the monotherapy group. There were no significant differences between the 2 active groups in any outcome measure, and both active groups experienced more adverse events than placebo. There were no systemic reactions. Improvement in multiple relevant outcomes strongly suggests that SLIT with timothy extract alone was effective; however, the results for symptom and medication scores were not significant. The differences between multiple allergen SLIT and placebo only in skin sensitivity to timothy suggest a reduction in SLIT efficacy in this group. However, further studies are required to confirm these observations.