Abstract A method for preparation of penicilloyl-polylysines by reaction of penicillin with polylysine at alkaline pH is described. The product is virtually free of penamaldate and penicillenate substituents, groups which are present when penicilloyl-polylysine is prepared from penicillenic acid. Penicilloyl-polylysines have been prepared from allylthiomethylpenicillin, methicillin, and phenethicillin as well as from bezylpenicillin. The penicilloyl-polylysines described above have been compared with respect to their ability to evoke cutaneous wheal-and-erythema responses in humans with penicillin allergy. Benzylpenicilloyl-polylysines prepared from penicillin and penicillenic acid are generally equivalent as elicitors of cutaneous responses. This indicates that the penicilloyl group is primarily responsible for allergic responses induced by penicilloyl-polylysines which also contain penamaldate and penicillenate substituents. In patients with benzylpenicillin allergy, there was a greater average cutaneous reactivity of benzylpenicilloyl-polylysine than of polylysines prepared from methicillin, phenethicillin, and allylthiomethylpenicillin. This finding suggests that the benzyl side-chain contributes to the antigen-antibody interaction. Steric hindrance by the side chains may also play a role in the lesser reactivity of penicilloyl-polylysines containing side groups other than benzyl.