With the aim of developing cytotoxic hybrid molecules which can be selectively targeted to specific cells in the gonads, a single chain ribosome-inactivating protein, gelonin, was conjugated to ovine luteinizing hormone (oLH) with the use of heterobifunctional cross-linking agents N-succinimidyl3-(2-pyridyldithio)-propionate (SPDP) and long-chain SPDP. Four hormonotoxins were synthesized having a variable spacer arm between oLH and gelonin. The spacer arms in C200A, C210A, C220A, and C230A were 13.6, 22.4, 22.4, and 31.2 A long, respectively. Extensive physiochemical and biochemical analysis revealed a 1:1 molar ratio of the ingredients in its oLH-S-S-gelonin conjugates. The linkage occurred through the epsilon-NH2 group of the alpha-subunit of oLH as judged from RP-HPLC analysis. The hormonotoxins retained substantial receptor binding ability, steriodogenic activity, and immunoreactivity of oLH and gelonin to their respective antibodies. Hormonotoxins bind to Leydig tumor cells via oLH, leaving gelonin free as judged by competitive displacement analysis. The hormonotoxins internalized to a sufficient degree to effectively inhibit protein synthesis. Upon comparison, immunoreactivity, receptor binding steroidogenic activity, and cytotoxicity of oLH-S-S-gelonin conjugates prepared with the use of only LC-SPDP (C230A, 31.2-A spacer arm) and by using both SPDP and LC-SPDP (C210A and C220A, 22.4-A spacer arm) were found to be comparable with that of conjugate prepared with SPDP alone (C200A, 13.6-A spacer arm). Therefore, it may be concluded that the cytotoxicity of oLH-based hormonotoxin remained unaffected with the use of long-chain spacer arms which are believed to be used generally to avoid steric hindrance.