Prostate cancer is a leading cause of death from cancer in American men and metastasis the main cause of death. To better understand the disease and accelerate development of new therapies, in vivo models that reflect different disease stages are needed. A family of cell lines that mimics multiple steps in cancer development and tumor progression has been developed in our laboratory from the parent, non-tumorigenic, RWPE-1 cell line by transformation with N-methyl-N-nitrosourea (MNU). The MNU cell lines mimic multiple steps in tumor progression where WPE1-NB26 is the most malignant cell line. WPE1-NB26 cells form metastases in the lungs of athymic, male, nude mice after intravenous injection. Two new cell lines, WPE1-NB26-64 and WPE1-NB26-65, showing more malignant characteristics than the parent WPE1-NB26 cell line, were derived from tumors after subcutaneous injection of WPE1-NB26 cells into nude mice. The WPE1-NB26-64 and WPE1-NB26-65 cell lines show an increase in anchorage-dependent growth and invasive ability as compared to the parent WPE1-NB26 cells. While the parent WPE1-NB26 cells express barely detectable levels, the new cell lines produce high levels of matrix metalloproteinase MMP-2 and detectable levels of MMP-9. By immunostaining, all three cell lines were positive for cytokeratins CK18 and CK5/14. These cell lines, having the same lineage, represent additional steps in the multi-step process of tumor progression and provide novel and useful cell models for studies on tumor progression and for drug development for the treatment of prostate cancer.