Purpose: We underwent this study to evaluate the factors which influence labeling efifciency when modified in vivo erythrocyte labeling technique was used. Materials and methods: Thirty healthy volunteers (M:F=19:11, age: 25¡¾2 yrs) were enrolled in this study. Totally, two hundred ten samples were obtained from them. The 1 mg of stannous pyrophosphate was injected intravenously at the beginning of labeling. After suitable tinning time (5 min, 20 min, 35 min) passed by, blood (5 mL, 3 mL or 1 mL) was withdrawn into 10 mL syringe previously containing Tc-99m (740 MBq) and anticoagulant (heparin, ACD or CPDA) through 19-gauged scalp needle. The generator ingrowth time of Tc-99m was within 24 hrs in each case. The blood samples were placed on rotating invertor during incubation (10 min, 25 min, 40 min) but some of them were not. Immediately after the conclusion of incubation, the labeled blood specimens to analyze were centrifuged. and then %Unbound Tc-99m was calculated. Statical analysis was used paired T-test and one way ANOVA with SPSS 10.0. Results: The binding efficiency at 1 mL of blood volume was 73¡¾32%, 91¡¾10% at 3 mL and 96¡¾7% at 5 mL (p<0.01). The binding efficiency at 5 min of tinning time was 45¡¾23%, 98¡¾6% at 20 min and 97¡¾8% at 35 min (p<0.001). The binding efficiency at 10 min of incubation time was 96¡¾7%, 95¡¾12% at 25 min and 98¡¾3% at 40 min (p>0.05). The binding efficiency in case of using rotating invertor was 96¡¾7% and the binding efficiency in case of not using it was 87¡¾18% (p>0.05). There was no significant difference between them. In binding efficiency according to kinds of anticoagulants, ACD was 98¡¾4%, CPDA was 97¡¾6% and heparin was 89¡¾20% (p<0.001). Conclusion: When modified in vivo erythrocyte labeling technique is used with Tc-99m, the methods to obtain the highest labeling efficiency are as follow. The withdrawing blood volume should be over 3 mL, tinning time should be kept between 20 min and 35 min, and incubation time should be kept between 10 min and 40 min. ACD or CPDA have to be used as a anticoagulant except heparin and the blood samples should be placed on rotating invertor during incubation. (Korean J Nucl Med 38(4):300-305, 2004)