Objectives: Endothelial cells harbor many antigenic determinants that may be targets for the immune system. The aim of this study was to determine the immunologic effects of decellularization, using 3 different methods, on xenograft rejection. Materials and Methods: In a sterile plate containing phosphate-buffered saline, fresh sheep aortic heart valves were decellularized using 3 different enzymatic methods: with 900 µg/mL of collagenase at 40°C (method A), with 450 µg/mL of collagenase at 4°C (method B), and with 900 µg/mL of collagenase at 4°C (method C). Intact and decellularized valves were implanted subdermally into inbred male albino rabbits and extracted after 21 days (extra valve pieces were also extracted after 60 days, as control samples, for assessing chronic rejection). Valves were histologically analyzed for inflammatory cell infiltration. Subendothelial structure integrity was determined using surface electron microscope. Results: No inflammatory cell infiltration was seen around the decellularized valve with method A, and no subendothelial structure change was observed by surface electron microscope. Infiltration of immune cells involved in rejection was not seen around valves decellularized with method B, although the subendothelial structure was relatively preserved and valve stiffness was increased. With method C, we observed a foreign body-type reaction around the intact valve and the decellularized valve. Conclusions: Method A is considered the optimal method of decellularization in our study, as this method significantly reduced the immune response to xenograft tissue, while maintaining subendothelial tissue.