To better understand androgen action in normal prostate cells, we characterized the androgen growth response of an immortalized nontumorigenic rat prostate cell line called CA25 that had been stably transfected with androgen receptor (AR) cDNA. Surprisingly, we found that AR(+) CA25 cells grew slower in the presence of dihydrotestosterone (DHT), whereas the growth of AR(−) CA25 cells was not affected by the hormone. DHT-mediated growth inhibition of CA25 cells was not attributable to an increase in apoptosis but rather to a morphological conversion consistent with terminal differentiation. Specifically, we found that DHT treatment of CA25 cells resulted in a striking change in cell architecture, localization of desmoplakin to cell-cell boundaries, and an increase in α6p integrin levels, a newly described marker of cell differentiation. Although no androgen-dependent changes were observed in the transcript levels of the mitochondrial aspartate aminotransferase or c-Myc genes by Northern blot analysis, RNA expression profiling of DHT-treated CA25 cells identified 282 genes of 1,018 that were continually expressed over a 48-h period. It was found that 63 of these genes were up-regulated >5-fold within the first 4 h of treatment and encoded functions involved in transport, signal transduction, and metabolism. These expression profile data are consistent with the striking morphological changes we observed in DHT-treated CA25 cells and provide a starting point for molecular analysis of in vitro prostate cell differentiation.