Various penicillin-based structures attached to paper disks were used in the radioallergosorbent test (RAST) to measure concentrations of circulating specific IgE antibodies in sera from subjects suspected of having penicillin allergy. The structures used were penicilloyl, penicillanyl, penicoyl and penicanyl groups prepared from benzylpenicillin (PG), phenoxymethylpenicillin (PV), amoxicillin (AX) and 6-amino-penicillanic acid (6-APA). The efficacy of these structures was evaluated using serum samples from 53 subjects who, on clinical grounds, were considered to have had adverse reactions to penicillins. The results showed the presence of IgE antibodies specific to the penicillin side-chain in certain patients, whereas others had IgE antibodies specific to the penicilloyl or penicillin basic structure (penicillin nucleus), which led to clinical cross-reactions between penicillins. The results showed that the use of several penicillin-based structures in a new test system increased the positive rate observed to 54.7% from 37.7% utilizing penicilloyl G, penicilloyl V and amoxicilloyl Phadebas RAST. Thus, the prepared species improve the capabilities of the in vitro diagnosis of penicillin allergy.