Three ricin A-chain immunotoxins (ITs) recognizing different antigens on Hodgkin-Reed/Sternberg (H-RS) cells were evaluated for their anti-tumor effects when used in combination as "cocktails". These ITs, BB10.dgA (CD25), HRS3.dgA (CD30), and IRac.dgA (70 kDa), strongly inhibited the growth of L540Cy H-RS cells in vitro. The protein synthesis of this cell line was reduced more efficiently by the combination of 2 of these ITs than by BB10.dgA, HRS3.dgA or IRac.dgA alone. A cocktail of all 3 ITs was most effective in vitro. This was at least in part due to the non-homogeneous distribution of CD25, CD30 or IRac on the L540Cy H-RS target cells and to the fact that subpopulations deficient in one antigen nevertheless expressed appreciable levels of the other target antigens. IT cocktails were also superior as anti-tumor agents in nude mice with solid L540Cy tumors. Ninety percent of mice treated with cocktails containing 2 or 3 ITs had continuous complete remissions (CCR), as compared with only 40% of mice treated with the same dose of a single IT. Analysis of 7 L540Cy sub-lines re-established ex vivo from mice that relapsed after having achieved complete remission (CR) after therapy with a single IT showed that the surviving tumor cells were antigen-deficient variants which were resistant to the original IT, but which could be killed by ITs directed against other target antigens. Thus, IT cocktails give superior results against human H-RS cells, both in vitro and in vivo.