To improve the in vivo stability of disulfide-linked immunotoxins (ITs), a series of sterically hindered cross-linking reagents were designed and synthesized. These ligands are characterized by a thioimidate group linked to an S-acetyl thiol or a substituted aryldithio group. To select the reagent of choice, several aryldithio thioimidates, substituted with a methyl or a phenyl group adjacent to the disulfide, were analyzed in thiol-disulfide exchange reactions. Also analyzed were the following: (i) the stability and solubility of the linkers in aqueous solution, (ii) the rate of protein derivatization, and (iii) the steric hindrance due to methyl or phenyl group substituents toward cleavage of the disulfide bond by glutathione. Ethyl S-acetyl 3-mercaptobutyrothioimidate (M-AMPT) was chosen as reagent to prepare two types of stable disulfide-containing AR-3-gelonin conjugates (IT2 and IT3). IT2 was prepared by a 3-(4-carboxamidophenyldithio)propionthioimidate (CDPT)-derivatized antibody coupled to the M-AMPT-derivatized gelonin to afford a conjugate characterized by the presence of a methyl group adjacent to the sulfide bond. In the IT3 conjugate, an M-AMPT-derivatized toxin was coupled to the antibody thiolated with M-AMPT and then activated with Ellman's reagent (DNTB). The in vitro and in vivo stabilities of the three immunoconjugates were assayed, respectively, (i) by adding an excess of glutathione and monitoring protein release and (ii) by studying their pharmacokinetic behaviors. The specificity and cytotoxicity of all ITs were analyzed on target and unrelated cell lines, and no significant differences in activity were observed. IT3, consisting of a symmetrical dimethyl-substituted disulfide bond, was substantially more stable in vivo (t1/2 beta = 88.3 h) than the corresponding IT2, characterized by a disulfide-protected monomethyl substituent bond (t1/2 beta = 60.2 h) compared to the unhindered conjugate IT1 (t1/2 beta = 27.9 h). This family of cross-linking reagents therefore offers advantages, such as minimal perturbation of the protein structure and controlled reactivity due to the thioimidate moiety, as well as the capacity to yield immunotoxins possessing substantial stability in vivo.