Since clinical phase-I/II trials in patients with resistant Hodgkin's lymphoma treated with the chemically linked anti-CD25 ricin-A-chain immunotoxin RFT5-SMPT-dgA indicate promising results for patients with minimal residual disease, we constructed a new immunotoxin by fusing the RFT5 single-chain variable fragment to a deletion mutant of Pseudomonas exotoxin A (ETA'). The recombinant protein was directed into the periplasmic space of E. coli by means of the pET-derived expression vector pBM1.1 and our newly developed expression/purification method. Biologically active RFT5(scFv)-ETA' was isolated by freezing/thawing and purified by immobilized metal-ion affinity and molecular-size-chromatography. RFT5(scFv)-ETA' was subsequently used for the treatment of disseminated human Hodgkin's lymphoma in a SCID-mouse model. The mean survival time (MST) of L540rec-challenged SCID mice was 38.1 days. A single i.v. injection of 40 microg recombinant immunotoxin (rIT) 1 day after tumor inoculation resulted in 100% tumor-free mice, extending the MST to more than 220 days (p < 0.0001). The blood-distribution time T(1/2)alpha was 39.65 min, the serum elimination time T(1/2)alpha, 756.6 min. All animals were assessed for soluble interleukin-2 receptor alpha, which is directly correlated to tumor burden. Soluble CD25 was not detectable in mice treated with the rIT. Our findings, concerning potent anti-tumor effects of a recombinant anti-CD25 immunotoxin against disseminated Hodgkin's lymphoma in SCID mice reported here demonstrate that RFT5(scFv)-ETA' might be suitable for further evaluation against Hodgkin's lymphoma in humans.