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Background Reliable closure of the gastrotomy after transgastric natural orifice transluminal endoscopic surgery (NOTES) remains unresolved. Objective To compare the technical aspects and clinical and histologic outcomes of NOTES gastrotomy closure techniques. Design Experimental study. Setting Animal laboratory. Patients Thirty-four dogs, 14 for nonsurvival study and 20 for survival study. Interventions The animals randomly received different gastrotomy closures after NOTES: endoclip, omentoplasty, over-the-scope-clip (OTSC), and hand-suturing. Main Outcome Measurements Procedure time, closure strength, survival, postoperative adverse events, and histologic evaluation of wound healing. Results Omentoplasty and OTSC groups needed shorter procedure times and fewer clips than the endoclip group. The endoclip and omentoplasty groups generated similar leakage pressures (34.5 ± 2.6 vs 42.2 ± 4.1 mm Hg, P > .05), both lower than OTSC and hand-suturing groups (81.5 ± 2.1 and 87.0 ± 3.0 mm Hg, respectively, P < .001). Of the 20 animals in the survival study (all 4 groups), only 2 of 6 in the endoclip group were killed prematurely due to sepsis. Necropsy revealed the OTSC group reached a 100% clip retention rate, higher than the endoclip (47.9%) and omentoplasty groups (44.4%, P < .05) rates. Complete healing, defined as intact and continuous gastric layers microscopically, was seen in 83.3% of animals (5 of 6) in the omentoplasty group, comparable with OTSC (4 of 6, 66.7%, P = .500) but higher than the endoclip group (1 of 6, 16.7%, P = .04). Limitations Animal study. Conclusions Omentoplasty is easier and safer for NOTES gastrotomy closure than endoclips and offers safety profile and efficacy similar to OTSC and hand-suturing.