Poly-L-ornithine with an average molecular weight of 32K was reacted with beta-amanitin hydroxysuccinimide ester to form an amide-linked toxin conjugate. Loading of the polymeric chain with amanitin was high, corresponding to up to 35% of the total weight. To this amatoxin vehicle we attached a targeting molecule, human recombinant leucine-21 epidermal growth factor (hrEGFL), via a disulfide-containing linker moiety. A typical average stoichiometry of the hrEGFL labeled toxin conjugate was (L-Orn)164(beta-amanitin)19(COC2H4SSC2H4CO-hrEGFL)2. The affinity for EGF receptors of hrEGFL bound in this conjugate was tested by using A 431 cells. The affinity was eight times lower than that of unsubstituted hrEGFL but regarded as high enough for studying specific toxicity effects with cells bearing EGF receptors. We found that beta-amanitin in the labeled conjugate was able to inhibit the growth of A 431 cells at a concentration of 28 nM, 80 times lower than for native beta-amanitin and 20 times lower than for poly-L-ornithine-bound beta-amanitin without the hrEGFL label. The approximately 20-fold enhancement of cytotoxicity suggests a specific internalization of the toxin conjugate mediated by the hormone label. This idea is supported by the fact that also in another transformed fibroblast cell line, with an increased though smaller number of EGF receptors than A 431 cells, the corresponding enhancement of cytotoxicity was demonstrable but less pronounced (7-fold). The hormone-mediated increase in cytotoxicity of EGF labeled poly-L-ornithine-beta-amanitin conjugates, combined with their moderate toxicity in the mouse, encourages further examination of such compounds in tumor model systems in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)