Objective:To establish and identify androgen-independent human prostate cancer cell line LNCap by culturing LNCaP cells with gradual deprivation of hormone.Methods: LNCaP cells were cultured in the medium with gradual deprivation of hormone (treated by active carbon to simulate androgen deprivation)for 10 days; and then the cells were cultured with complete deprivation of androgen for 3 months till the cell entered the rapid proliferation phase again.The cell growth and expression of PSA and androgen were examined by CCK-8,immunfluorescence and RT-PCR methods.Results: LNCaP cells grew slowly after deprivation of hormone and took on a neuroendocrine phenotype and cluster growth pattern.After 3 months’ non-androgen culture,the cells regained original morphology and growth.CCK-8 indicated that LNCaP cells could grow in non-androgen condition; immunofluorescence assay indicated that LNCaP-AI cells could regain PSA-secreting activity in non-androgen condition; and RT-PCR suggested that androgen was highly expressed in LNCaP-AI cells.Conclusion: Androgen-independent LNCaP cell line can be established by culturing with gradual deprivation of hormone for 3 months.