A cloned cell line (R3327H-G8-A1) has been isolated from the Dunning R3327H adenocarcinoma. Light and electron microscopic studies showed that the cell line possessed features common to secretory epithelial cells. These cells, which grow in monolayer culture, produced s.c. hind flank tumors when inoculated inoculated into Copenhagen X Fischer F1 rats.l Chromosomal karyotype analysis confirmed that the cell line is distinctly that of the Rattus norvegicus genus and species. The cells specifically bind testosterone and dexamethasone with equilibrium dissociation constants (Kd) of 0.49 and 0.8 nM, respectively. The numbers of saturable binding sites per cell are 10,000 for testosterone and 60,000 for dexamethasone. The cells also have 5 alpha-reductase activity. These properties are characteristic of the prostate and of the Dunning tumor from which the cells are derived. Cell growth in vitro was stimulated by androgens and inhibited by glucocorticoids at concentrations of 10(-8) M. An int riguing finding was that estradiol and progestins dramatically stimulated growth in the apparent absence of receptors for these hormones. Finally, comparisons between the G8-A1 cells and the tumor induced by the G8-A1 clone and a second generation of cells from this G8-A1-induced tumor showed that the cloned cells retained their properties following passaging in the animal.