We sought to evaluate the feasibility of the retroperitoneum's exploration via natural orifice transluminal endoscopic surgery (NOTES) using transvaginal access in a porcine model, and its possible application in human beings. Six female pigs (25-30 kg) were used to establish anatomic landmarks and technical steps. Six additional pigs were used for the survival study. Under general anesthesia and with the pig supine, a 1-cm posterolateral colpotomy was performed with the double-channel gastroscope's needle knife. The incision was enlarged laterally using blunt dissection while keeping in contact with the psoas muscle. A retroperitoneal tunnel was created using carbon-dioxide dissection and the movements of the gastroscope up to the level of the kidney. The colpotomy site was closed using interrupted sutures (polyglactin 2/0). A follow-up laparoscopy and necropsy were performed 3 weeks postoperatively. Successful access to the retroperitoneum was achieved in all pigs with a mean operative time of 30 minutes. However, in the first 3 pigs used for the acute study, the peritoneum was perforated during the six-pig dissection and the procedure was abandoned because of the space's collapse. No perforation occurred during the survival study. An excellent view of the retroperitoneal space and structures, such as the vascular and lymphatic tissues, the kidney, the adrenal gland, and the ureter, was obtained. No intraoperative complications or bleeding or injury to any of the retroperitoneal structures occurred. The 3-week follow-up laparoscopy showed no adhesions or abscesses. These findings were confirmed at necropsy. The retroperitoneal space can be successfully accessed via NOTES. Transvaginal NOTES access to the retroperitoneum avoids any transparietal trocars, so it could decrease surgical trauma, be better tolerated, and offer better visualization, with the obvious gender limitation. Future clinical application in human beings may include procedures such as lymphadenectomy, nephrectomy, and adrenalectomy.