Purpose: Transvaginal cholecystectomy with laparoscopic assistance has been performed safely in humans. Cholecystectomy with Natural Orifice Translumenal Endoscopic Surgery (NOTES) without laparoscopic support will require new techniques and instruments for access, gallbladder retraction, dissection, and sealing of the cystic duct and artery. The aim was to develop a NOTES technique to perform transvaginal cholecystectomy without laparoscopic instruments using prototype flexible endoscopic devices. Methods: Cholecystectomies were performed in 88-97 lbs pigs with a planned two week survival. Prototype flexible instruments (NOTES Toolbox 1 provided by Ethicon Endo-Surgery Inc.) included a steerable overtube, bipolar hemostatic forceps, Maryland dissector, clip applier, grasper, rotating hook knife, scissor, and a tissue approximating system (TAS). A dual channel endoscope was passed transvaginally and pneumoperitoneum was established. Endoscope stabilization was achieved with the steerable trocar or a suture through a working channel of the endoscope and the abdominal wall. Sutures, endo-loops, or the TAS were attached to the gallbladder and externalized through the abdominal wall with a 1 mm suture passer for retraction. Dissection in the triangle of Calot to achieve the critical view was performed with endoscopic scissor, hook knife, or Maryland. The cystic duct was sealed with prototype clips and the artery was clipped or cauterized. The gallbladder was dissected off the liver with scissor and hook knife. The gallbladder was removed from the vagina under direct vision. The colpotomy was closed with 3-0 absorbable sutures.Results: Three out of the five pigs survived two weeks; two died early from liver bed hemorrhage. Operative times ranged from 115-275 minutes and the operative times decreased with experience. The critical view was established in all cases. Perforation of the gallbladder occurred in four pigs. At necropsy, all clips placed on the cystic duct were secure. The artery was secure when clipped in two or coagulated in three pigs. There was no evidence of bile leak or infection. Conclusions: NOTES cholecystectomy without laparoscopic support is feasible using prototype flexible endoscopic devices. The transvaginal steerable trocar gives consistent access to the gallbladder. Complete gallbladder dissection is possible and the clip applier is effective in controlling the cystic duct. The cystic artery can be controlled with clip or coagulation. The primary remaining difficulty is lack of precision during dissection. Techniques and instrumentation for pure NOTES cholecystectomy have improved, but further refinement is warranted.