The purpose of this study is to develop an initial step in salivary gland tissue engineering through proliferation and phenotypic preservation of rat parotid acinar cells in vitro. By using the explant outgrowth technique and M199 medium with the addition of sialic acid, acinar cells not only survived for more than 30 days in the absence of basement membrane substrates but also proliferated to yield cells with acinar phenotypic expression. Furthermore, we tested whether chitosan can be used as a synthetic extracellular matrix to culture salivary acinar cells. Chitosan is a deacetylated product of chitin, which is a plentiful polysaccharide found in nature and is safe for the human body, but little is known about the utility of chitosan in culturing salivary acinar cells. It was found that coating fibronectin on chitosan membrane improved the attachment of acinar cells in the initial stage. However, the poor attachment of acinar cells on pure chitosan membrane did not affect cell growth after longer culture times, indicating that chitosan is potentially useful as a tissue-engineering scaffold of the salivary gland. These in vitro results are encouraging because such a culture system may serve as an artificial salivary gland for future use in the treatment of patients with salivary hypofunction.