Secretions of salivary glands are essential for the maintenance of oral health. Due to the lack of suitable in vitro models, studies to examine biochemical and molecular mechanisms of the cellular secretions have been difficult. Furthermore, adequate quantities of human epithelial cells could not be obtained, because normal diploid cells are believed to exhibit a limited lifespan of two to three passages (40-50 population doublings). This report describes for the first time the development of two diploid epithelial acinar cell lines, HPAM1 and HPAF1, derived from the normal human parotid gland. The cell lines are propagated in serum-free medium comprised of keratinocyte basal medium supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 micrograms/ml), epidermal growth factor (EGF, 10 ng/ml), bovine pituitary extract (25 micrograms/ml), and antibiotics. The HPAM1 cell line has been passaged more than 50 times (> 189 population doublings) and HPAF1 more than 40 times (> 185 population doublings). Both cell lines exhibit normal diploid karyotypes, lack transformed phenotypes and are non-tumorigenic in nude mice. Both cell lines produce tissue-specific proteins, i.e. alpha-amylase 1, basic proline-rich protein, and cystatins; and express the corresponding genes as determined by RT-PCR analyses. These results demonstrate that normal diploid human cells do not inherently exhibit limited life-span in vitro and can, under optimum conditions, be propagated indefinitely.