Denture stomatitis (DS) is a fungal infection characterized by inflammation of the oral mucosa in direct contact with the denture and affects up to 50% of denture wearers. Despite the prevalence, very little is known about the role of fungal or host factors that contribute to pathogenesis. Recently, we developed a novel intraoral denture system for rodent research. This denture system consists of custom-fitted fixed and removable parts to allow repeated sampling and longitudinal studies. The purpose of this study was to use this denture system to develop a clinically relevant animal model of DS. To establish DS, rats were inoculated with pelleted Candida albicans, which resulted in sustained colonization of the denture and palate for 8 weeks postinoculation. Biofilm formation on the denture was observed by week 4 and on the palate by week 6 postinoculation. Rats were monitored for clinical signs of disease by assigning a clinical score after macroscopic examination of the palate tissue according to Newton's method. By week 4 postinoculation, the majority of inoculated rats with dentures exhibited a clinical score of 1 (pinpoint erythema). By week 6 and week 8 postinoculation, increasing percentages of rats exhibited a clinical score of 2 (diffuse erythema/edema). Histological analysis of palate tissue demonstrated progressively increasing inflammatory cell recruitment throughout the time course of the infection. Palatal biofilm formation was commensurate with development of palatal erythema, which suggests a role for biofilm in the inflammatory response.