Pseudomonas exotoxin A (PE) is a three-domain protein in which domain Ia is involved in recognition of receptors on eukaryotic target cells, domain II promotes translocation of PE into the cytosol, and domain III enzymatically ADP-ribosylates elongation factor 2. Modification of proteins with polyethylene glycol (PEG) has been shown to prolong circulating plasma lifetime and may reduce or eliminate immunogenicity. However, in the case of toxins, PEG may interfere with or block toxin activity. To investigate the effect of polyethylene glycolation on specific residues located on the surface of PE domain II, we substituted cysteine, for each of the five most exposed surface amino acids (H276, E282, N306, R313, and E327) in domain II. These cysteines can serve as unique sites for PEG modification. The PE-Cys proteins retained most of their cytotoxicity even when the free sulfhydryl group was blocked by 5,5'-dithiobis(nitrobenzoic acid) or glutathione. When the PE-Cys proteins were conjugated with ovalbumin using a cleavable disulfide linkage, cytotoxicity was retained, but it was lost with a non-cleavable thioether linkage. In contrast, cytotoxicity was maintained when PE-Cys mutants were coupled to 5- or 20-kDa mPEG, using either a disulfide or a thioether linkage. Unexpectedly in some cases, the thioether conjugate was more active than the disulfide linkage. Pharmacokinetic studies on one of the polyethylene-glycolated molecules (R313C) showed that the mean residence time (t 1/2) was prolonged to 72 min, compared to 20 min for unpolyethylene glycolated PE-Cys(R313C). These studies show it is possible to derivatize PE at specific residues in domain II, maintain significant cytotoxic activity, and alter pharmacokinetics. These studies also suggest that large mPEG molecules can be translocated to the cytosol while still attached to domain II of PE.