Ambrosia artemisiifolia L. is a leading cause of allergies in many countries. In the last decade it has appeared and spread in Bulgaria as well. The aims of this study was to collect pollen from ragweed plants in Bulgaria, prepare the first Bulgarian ragweed allergen, and characterize it. The new allergen was studied in parallel with two other ragweed pollen extracts from the USA and Russia. The protein profile was studied through isoelectric focusing, and the total allergen activity was studied by a skin prick test and a basophil activation test. The proteins of the studied extracts were concentrated in the pI region 3.5–5.5 with an identical band number and location. The allergens showed similar total allergen activity in vivo:14/81 tested patients (17.3 %) were SPT positive either to a single ragweed extract or to all three. The allergens had identical histogram profiles and caused basophil degranulation above the test cut-off. The analysis of the flow cytometry results by parallel line bioassay shows linear dose-response relation between the extracts. The observed immunological properties of the Bulgarian allergen from Ambrosia artemisiifolia provide a possibility to use this product for a reliable diagnosis and effective specific immunotherapy of ragweed allergy in our country.