SummaryThe role of three penicillin metabolites in human allergic reaction to penicillin has been assessed using sensitivein vitrotests in addition to skin testing. Conjugates of the benzylpenicilloyl (BPO), benzylpenicillenate (BPE) and penicillamine (PA) groups with human serum albumin (HSA) and bovine gamma globulin (BOG) have been made. In the passive haemagglutination test antibodies specific for all three determinants were found but there was no difference between allergic and non‐allergic groups. However, the ratio IgG/IgM, specific for each determinant, was higher in normals than in allergies. In skin tests, histamine release from leucocytes and the lymphocyte transformation test, the BPO group was confirmed as the major determinant, but in some individuals the BPE or PA groups are more importan