We have developed a novel methodology for site-specific pegylation of proteins by use of transglutaminase (TGase). In this methodology, alkylamine derivatives of poly(ethyleneglycol) (PEG) could be site-specifically incorporated into intact or chimeric proteins without decreasing the bioactivities. The incorporation site of the TGase-catalyzed modification is limited to the substrate Gln residues for TGases. The high homogeneity of the constructed conjugates and the ability to design conjugates with suitable incorporation sites will improve the applicability of PEG-protein conjugates for clinical use.