DNA　repair　enzyme　O-6-methylguanine-DNA　methyltransferase　(MGMT),　which　plays　an　important　role　in　inducing　drug　resistance　against　alkylating　agents　that　modify　the　O-6　position　of　guanine　in　DNA,　is　an　attractive　target　for　anti-tumor　chemotherapy.　A　series　of　MGMT　inhibitors　have　been　synthesized　over　the　past　decades　to　improve　the　chemotherapeutic　effects　of　O-6-alkylating　agents.　In　the　present　study,　we　performed　a　three-dimensional　quantitative　structure　activity　relationship　(3D-QSAR)　study　on　97　guanine　derivatives　as　MGMT　inhibitors　using　comparative　molecular　field　analysis　(CoMFA)　and　comparative　molecular　similarity　indices　analysis　(CoMSIA)　methods.　Three　different　alignment　methods　(ligand-based,　DFT　optimization-based　and　docking-based　alignment)　were　employed　to　develop　reliable　3D-QSAR　models.　Statistical　parameters　derived　from　the　models　using　the　above　three　alignment　methods　showed　that　the　ligand-based　CoMFA　(Q(cv)(2)　=　0.672　and　R-ncv(2)　=　0.997)　and　CoMSIA　(Q(cv)(2)　=　0.703　and　R-ncv(2)　=　0.946)　models　were　better　than　the　other　two　alignment　methods-based　CoMFA　and　CoMSIA　models.　The　two　ligand-based　models　were　further　confirmed　by　an　external　test-set　validation　and　a　Y-randomization　examination.　The　ligand-based　CoMFA　model　(Q(ext)(2)　=　0.691,　R-pred(2)　=　0.738　and　slope　k　=　0.91)　was　observed　with　acceptable　external　test-set　validation　values　rather　than　the　CoMSIA　model　(Q(ext)(2)　=　0.307,　R-pred(2)　=　0.4　and　slope　k　=　0.719).　Docking　studies　were　carried　out　to　predict　the　binding　modes　of　the　inhibitors　with　MGMT.　The　results　indicated　that　the　obtained　binding　interactions　were　consistent　with　the　3D　contour　maps.　Overall,　the　combined　results　of　the　3D-QSAR　and　the　docking　obtained　in　this　study　provide　an　insight　into　the　understanding　of　the　interactions　between　guanine　derivatives　and　MGMT　protein,　which　will　assist　in　designing　novel　MGMT　inhibitors　with　desired　activity.