BACKGROUND The available human prostate cancer cell lines that are metastatic in athymic nude mice all have complex, highly aneuploid karyotypes. Other prostatic cells immortalized by transforming genes of SV40 or HPV and converted to tumorigenicity by additional genetic manipulation are not reported to be metastatic. METHODS Tumorigenic sublines of human prostate epithelial cells previously immortalized by transfection with the SV40T antigen gene were obtained by sequential passage in male athymic nude mice. These sublines were evaluated histopathologically for tumorigenicity and metastasis in athymic nude mice after subcutaneous, intraperitoneal, and intraprostatic injection. Each subline was characterized by standard (GTG-banding) cytogenetic and FISH analysis, and RNase protection assays for androgen receptor expression. RESULTS Two sublines produced metastases in lungs and the diaphragm of most mice after either intraprostatic or intraperitoneal injection. The M2205 subline formed large local tumors after intraprostatic injection. Cytogenetic aberrations present in the metastatic sublines, but not in the tumorigenic, nonmetastatic lines or the parental P69SV40T line, included dup(11)(q14q22), der(16) t (16;19) (q24;q13.1), which resulted in the loss of the short arm and proximal long arm of chromosome 19 (19q13.1-->19pter), and loss of the Y chromosome. None of the sublines expressed the androgen receptor. CONCLUSIONS These cytogenetically defined, SV40T-immortalized human prostate epithelial cell lines, with distinct biological behaviors in vivo, provide additional tools for the genetic analysis of the emergence of metastatic capacity.