BACKGROUND: Immediate allergic reactions to β-lactam antibiotics are mediated by specific IgE antibodies. The Phadia CAP System FEIA® is a commercial method for quantification of specific IgE. We wished to determine anti-β-lactam IgE antibodies in patients without penicillin allergy but with high levels of total IgE. METHODS: Sera from 41 patients (31 with high total IgE, 10 with low total IgE) were analyzed for IgE antibodies specific to penicilloyl G, penicilloyl V, amoxicilloyl and ampicilloyl using the CAP FEIA® method that was available up to 2006. Seven sera that tested positive were rechecked in a new improved system available after 2006. RESULTS: In patients without a history of penicillin allergy, the specificities of commercial tests for anti-β-lactam IgE antibodies were 100%, 60%, 27% and 20% at total IgE levels of 8–263 kU/l, 500–664 kU/l, 1000–2000 kU/l and > 2000 kU/l, respectively. In seven retested sera, only 2 (28%) were still positive for penicillin-specific IgE antibody. CONCLUSION: Before 2006, tests for anti-β-lactam IgE antibody in patients with total IgE > 500 kU/l were probably often false positive. Patients who were diagnosed as penicillin allergic before 2006 solely on the basis of a positive CAP FEIA® test for specific IgE should be considered for diagnostic reevaluation.