Radiolabeling of proteins is a widely used approach to study their in vivo disposition patterns. However, the obtained results may largely depend on the radiolabeling method used. The purpose of the present study was to investigate the effect of the radiolabeling method on the pharmacokinetic analysis of liver targeted protein in mice. Galactosylated bovine serum albumin (Gal-BSA) was labeled with 125I or 111In, using diethylenetriamine-pentaacetic dianhydride (cDTPA) or 1-(4-isothiocyanobenzyl)ethylenediaminetetraacetic acid (SCN-Bz-EDTA) as bifunctional chelating agents. The Gal-BSA was then injected in mice by a bolus intravenous injection. Samples of plasma, urine, liver, kidney, intestine and feces were collected at various time intervals and their radioactivity was measured. In none of the samples examined was there any significant difference in radioactivity distribution originating from the radiolabeling methods within 5 min after administration. After this period, 125I radioactivity in the liver started to decrease significantly faster than that of 111In, which would indicate the intracellular degradation of the protein. Consequently, the reappearance of trichloracetic acid (TCA) soluble 125I radioactivity in the plasma occurred. But whereas the hepatic uptake clearance (CLliver) of [111In]DTPA-Gal-BSA remained constant during 8 h postinjection, the CLliver of [125I]Gal-BSA at 30 min represented only one eighth of its initial values. The CLliver of [111In]SCN-Bz-EDTA-Gal-BSA resembled that of [111In]DTPA-Gal-BSA within 1 h of the experiment but it started to decline after this interval. The observed discrepancies most probably resulted from the formation of different radiolabeled metabolites in the hepatocytes and their different capability of crossing biological membranes. Our findings indicate that among the three methods employed, [111In]DTPA radiolabeling of Gal-BSA is the most appropriate method to study its tissue disposition.