Background Serum IgE antibodies directed at galactose‐α‐1,3‐galactose (α‐Gal) are associated with a novel form of delayed anaphylaxis occurring upon consumption of red meat or innards. Pork kidney is known as the most potent trigger of this syndrome, but the culprit allergens have not yet been identified. The aim of this study was the identification and characterization of pork kidney proteins mediating delayed anaphylactic reactions through specific IgE to α‐Gal. Methods A cohort of 59 patients with specific IgE to α‐Gal was screened by immunoblot for IgE‐reactive proteins in pork kidney. Proteins were identified by peptide mass fingerprinting. Isolated proteins were assayed in ELISA and ELISA inhibition, basophil activation and skin prick test. Results Several IgE‐binding proteins of high molecular weight (100– >200 kDa) were detected in pork kidney extracts by immunoblot using patient sera and an anti‐α‐Gal antibody. Two major IgE‐binding proteins were identified as porcine angiotensin‐I‐converting enzyme (ACE I) and aminopeptidase N (AP‐N). Reactivity of patient sera and anti‐α‐Gal antibody to both proteins was abolished by carbohydrate oxidation. The α‐Gal IgE epitopes were resistant to heat denaturation. Pork kidney extract, isolated ACE I, and AP‐N were able to activate patient basophils and elicit positive responses in skin prick tests. Conclusion Two cell‐membrane proteins carrying α‐Gal epitopes were identified in pork kidney. For the first time, isolated meat proteins were shown to induce basophil activation in patients with delayed anaphylaxis to red meat providing further confirmation for the clinical relevance of these α‐Gal‐carrying proteins.