We developed and characterized an immortalized rat parotid cell line to use in salivary gland studies. The cells were immortalized by retroviral transduction of SV40 large T antigen into isolated parotid cells. Using immunocytochemical techniques, we found that the immortalized epithelial cells were ductal, rather than acinar, in nature. Cells were grown under coculture conditions with lethally irradiated NIH3T3 cells. One cell line, which was designated RPG1/SV40 cells (for rat parotid gland 1/SV40 transformant), was selected for characterization. These cells formed a sheet epithelium with tight junctions and a measurable transepithelial resistance. RPG1/SV40 cells responded to muscarinic receptor (carbachol) and/or P2 purinoceptor (ATP and UTP) stimuli with increases in the following: (1) intracellular free-calcium concentration ([Ca2+]i); (2) the short-circuit current (ISC) across the epithelium; (3) the tyrosine phosphorylation of PKCδ; and (4) MAP kinase activity. Thus, the cells appear to be useful for a wide range of studies involving physiology, biochemistry, and signal transduction approaches.