We have established two new epithelial cell lines (NRP-152, NRP-154), with markedly different properties, from the dorsal-lateral prostate of Lobund/Wistar rats treated with N-methyl-N-nitrosourea and testosterone propionate. NRP-152 cells do not form tumors in athymic mice and retain many of the properties of normal prostatic epithelial cells. They produce prostatic acid phosphatase, have functional androgen receptors, and require the combination of several growth factors in addition to serum for optimal growth. Their growth is stimulated by epidermal growth factor, insulin, dexamethasone, cholera toxin, dihydrotestosterone, and testosterone, and their growth is inhibited by transforming growth factor βs and retinoic acid. These cells also respond to 1,25-dihydroxy-vitamin D₃ with an early growth stimulation followed by growth inhibition at later times. In contrast, tumorigenic NRP-154 cells lack detectable androgen receptor mRNA and have less stringent growth factor requirements for optimal growth. Growth of NRP-154 cells is stimulated by dexamethasone and insulin, inhibited by transforming growth factor β1, but not significantly altered by epidermal growth factor, cholera toxin, dihydrotestosterone, retinoic acid, or 1α,25-dihydroxyvitamin D₃. Our data suggest that the NRP-152 and NRP-154 cell lines are suitable systems for analysis of normal prostate growth and prostatic carcinogenesis.