The owl monkey (Aotus nancymae) is a primate with a bile acid and biliary lipid profile resembling that of humans. Aotus spp. are among the rare species, including humans, that spontaneously develop cholesterol gallstones. With dietary induction the owl monkey proved a rapid, reliable model of cholesterol cholelithiasis. Six owl monkeys, three of each sex, were fed a diet supplemented with 1.5% cholesterol for 5 weeks. Each week blood samples were drawn for cholesterol determination, and bile samples were obtained by ultrasound-guided percutaneous aspiration of the gallbladder. Weekly ultrasound imaging documented development of gallbladder sludge in all animals, with eventual stone formation in five of six. At necropsy after 5 weeks consuming the diet, all animals had distinct sludging and/or small stones in the gallbladder, correlating with the ultrasound findings. Plasma cholesterol values remained lower in females but increased markedly in some males to > 1,400 mg/dl. Histologic examination revealed mild, diffuse hepatocellular lipidosis and degeneration in four of six animals. Detailed examination of the gallbladder indicated that transhepatic needle punctures induced minimal focal abnormalities, judged inconsequential. In contrast to rodent models commonly in use, owl monkeys have liver and digestive tract anatomy and bile physiology that is similar to that in humans. These similarities give this model the potential to substantively improve understanding of the pathogenesis, treatment, and prevention of human cholesterol gallstones. This model can provide sequential, simultaneous correlation of plasma and biliary lipids, imaging of gallbladder contents, and physiologic processes.