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Background: In Europe, allergenic sensitization to ambrosia has become of increasing importance during the last years. Objective: To determine the prevalence of sensitization to ambrosia and its clinical impact in adults in East Germany. Methods and patients: Subjects were recruited from the population-based LIFE-Adult-Study in Leipzig. 2,626/4,088 (64.2%) had valid skin prick test (SPT) and were included. At recall, selected subjects (n = 70) with previously positive SPT to ragweed underwent repeated SPT and nasal provocation test (NPT)/anterior rhinomanometry (AR). When NPT/AR was valid, spirometry, sIgE and basophil activation test (BAT) were performed. Results: History was positive in 29.7% for allergic rhinitis/AR, in 8.0% for asthma. 111/2,626 (4.2%) investigated had positive SPT to ambrosia and 254 (9.7%) to mugwort. At recall, SPT was positive in 84.3% for ambrosia and in 75.7% for mugwort (cosensitization 86.4%). NPT/AR was valid in 45/70 (64.3%) and positive in 20/45 (44.4%). In this group, 22/45 (48.9%) reported respiratory symptoms during end of summer time. sIgE was positive for Ambrosia (A.) artemisiifolia in 25/45 (55.6%), mugwort 29/45 (64.4%), Amb a 1 5/45 (11.1%). Conclusion: This is the first study to evaluate the prevalence of ambrosia sensitization in adults in East Germany. SPT to ambrosia was positive in 4.2%. Frequent positivity of NPT/AR may indicate the clinical relevance of this sensitization.