Initial manifestation of composite tissue allograft (CTA) rejection is reflected at the microcirculatory level. Intravital microscopy provides invaluable information regarding the mechanism of allograft rejection and the outcome of therapeutic modalities. With the development of variety of inbred, recombinant, and mutant mice strains, a greater biological understanding of allograft rejection is possible, however the implementation of a mouse model in CTA transplantation studies is challenging due to the technical difficulties. The purpose of this study was to develop a CTA transplantation model in mice combining the advantages of intravital microscopy with the access to transgenic mice technology. The mice cremaster muscle transplantation model was developed and tested in the following experimental groups. Isograft control transplantations (n=6) were performed between C57BL/6N mouse. In the allograft group (n=5), transplantations were performed across fully mismatched strains between C3H and C57BL/6N mouse. The donor cremaster muscle allografts were dissected as a free flap and harvested on the common iliac artery and vein, and anastomosed to the recipient’s ipsilateral carotid artery and the external jugular vein using standard end-to-end technique (11/0 Ethilon under 40X magnification). After clamp release, cremaster muscle flap was prepared for direct in vivo observation of the microcirculation. The following measurements were taken during 1st and 2nd hour after transplantation: red blood cell velocities (RBC) and vessel diameters of the 1st, 2nd,3rd order arterioles; functional capillary perfusion (FCP); the number of rolling, adhering and transmigrating polymorphonuclear leukocytes and lymphocytes in the postcapillary venule; and endothelial edema index. The average time for cremaster transplantation was 2 hours and the average ischemia time was 75 minutes with a 95% success rate. RBC velocity and vessel diameters showed a normal pattern during the first 2 hours without significant differences between the groups. However, leukocyte-endothelial interaction were activated in the allograft group. The number of rolling (70.3±14), adhering (44.2±2.9) and transmigrating (7±1.5) leukocytes were increased significantly when compared to isograft control group (Rolling:14.8±2.2, Adhering:4.2±0.4, Transmigrating:1.96±1.3, p<0.001). Significant activation of the rolling and adhering lymphocytes was also observed in the allograft group (Rolling:5.6.±0.6, Adhering:4.6±0.9) when compared to isograft controls (Rolling:0.63±0.1, Adhering:0.66±0.3, p<0.05). This study proved the feasibility to transplant cremaster muscle flap in mice with 95% success rate. To our knowledge, this is the first report on composite tissue transplantation model, which offers the possibility to combine transgenic mice technology with direct in vivo evaluation of transplant hemodynamics via intravital microscopy system.