Recently, we created a lysine-deficient mutant tumor necrosis factor-alpha [mTNF-alpha-Lys(-)] with full bioactivity in vitro compared with wild-type TNF-alpha (wTNF-alpha), and site-specific PEGylation of mTNF-alpha-Lys(-) was found to selectively enhance its in vivo antitumor activity. In this study, we attempted to optimize this PEGylation of mTNF-alpha-Lys(-) to further improve its therapeutic potency. mTNF-alpha-Lys(-) was site-specifically modified at its N-terminus with linear polyethylene glycol (LPEG) or branched PEG (BPEG). While randomly mono-PEGylated wTNF-alpha(ran-LPEG(5K)-wTNF-alpha) with 5 kDa of LPEG (LPEG(5K)) had about only 4% in vitro bioactivity of wTNF-a, mono-PEGylated mTNF-alpha-Lys(-) [sp-PEG-mTNF-alpha-Lys(-)] with LPEG(5K), LPEG(20K), BPEG(10K), and BPEG(40K) had 82%, 58%, 93%, and 65% bioactivities of mTNF-alpha-Lys(-), respectively. sp-LPEG-mTNF-alpha-Lys(-) and sp-BPEGIOKmTNF-alpha-Lys(-) had much superior antitumor activity to those of both unmodified TNF-alphas and ran-LPEG(5K)-wTNF-alpha, though sp-BPEG(40K)-mTNF-alpha-Lys(-) did not show in vivo antitumor activity. Thus, the molecular shape and weight of PEG may strongly influence the in vivo antitumor activity of sp-PEG-mTNF-a-Lys(-). alpha 2004 Elsevier Inc. All rights reserved.