In our previous microarray analysis searching for genes differentially regulated by androgens in the rat ventral prostate, we identified GADD45gamma (growth arrest and DNA damage inducible, gamma) as one of the genes up-regulated by androgens. GADD45gamma was initially identified to be a gene involved in negative growth control and its overexpression induced cycle arrest and apoptosis in vitro. In this study, we showed that GADD45gamma was transiently up-regulated by androgens in the androgen-responsive human prostate cancer cell line LNCaP. The GADD45gamma up-regulation was blocked by an androgen receptor (AR) antagonist, bicalutamide, suggesting the involvement of the androgen receptor. However, this up-regulation was inhibited by cycloheximide, indicating that GADD45gamma induction by androgens requires new protein synthesis. Overexpression of GADD45gamma inhibited cell growth of LNCaP and PC3 cells and resulted in dramatic morphological changes in both cell lines, arguing that GADD45gamma is likely to participate in the differentiation program induced by androgens in the prostate. The above observations provide evidence that GADD45gamma is an androgen-responsive gene with growth-inhibitory activity in human prostate cancer cells.