The study of lacrimal dysfunction and insufficiency, a major cause of dry eye, has been hampered by the inability to induce the proliferation of primary lacrimal acinar cells in vitro. Particularly in light of observations that androgens are able to support the overall size and functional status of the lacrimal glands as well as certain specific lacrimal functions, an in vitro culture system that is permissive for cell proliferation would be most beneficial to study the molecular basis for these processes. Here, we report on the successful establishment of such a system. Using a culture system containing Hepato Stim Medium and Matrigel, we were able to induce the efficient proliferation of primary rabbit lacrimal gland acinar cells with epidermal growth factor (EGF) and dihydrotestosterone (DHT). The generation of this in vitro cell culture system should greatly facilitate study of the regulation of acinar cell function at the molecular and cellular levels.