Background Dog dander is an important cause of respiratory allergy, but the spectrum of known dog allergens appears incomplete. Two lipocalins, Can f 1 and Can f 2, and serum albumin, Can f 3, have been characterized in detail but do not fully account for the IgE antibody-binding activity of dog dander extract. Allergen activity has previously been detected in dog urine but not further characterized. Objective We sought to identify, characterize, and assess the importance of allergen components in dog urine. Methods Dog urine was fractionated by means of size exclusion chromatography and examined for IgE antibody binding. A protein present in one fraction displaying IgE antibody-binding activity was identified by means of N-terminal sequencing and mass spectrometry. A recombinant form of the protein was produced in Pichia pastoris . IgE antibody binding to dog allergen components among sera of 37 subjects with dog allergy was determined by means of ImmunoCAP analysis. Results An IgE antibody-binding protein was isolated from dog urine and identified as prostatic kallikrein. A closely related or identical protein was detected in dog dander. The recombinant prostatic kallikrein displayed immunologic and biochemical properties similar to those of the natural protein and bound IgE antibodies from 26 (70%) of 37 sera of subjects with dog allergy, 14 of which reacted to none of Can f 1, Can f 2, or Can f 3. The dog allergen identified here was found to cross-react with human prostate-specific antigen, a key culprit in IgE-mediated vaginal reactions to semen. Conclusion Prostatic kallikrein is a new major dog allergen.