A series of functional studies were performed to assess the potential role of the ras-related transformation suppressor gene, Krev-1, in suppressing prostate cancer cell growth. Three human prostate cancer cell lines, PC-3, TSU-Pr1, and DU-145 were transfected with a plasmid containing the Krev-1 cDNA and a neomycin resistance gene. Selected G418-resistant clones were isolated and expanded into cell lines. All cloned transfectants exhibited a significant reduction in their in vitro growth rates, i.e., longer doubling times, when compared to the parental cell lines. Molecular analysis of the Krev-1 cloned transfectants revealed that they all contained variable copy numbers of the Krev-1 gene and expressed high levels of Krev-1 mRNA transcript, as shown by Southern and Northern analysis, respectively. To determine whether the biological properties associated with tumorigenicity were changed in these Krev-1 transfectants, their growth characteristics were examined on the basis of their ability to a) form colonies in soft agar, and b) produce tumors in SCID mice. The majority of the Krev-1 transfectants from the PC-3 and TSU-Pr1 cell lines showed a substantially reduced ability to form colonies in soft agar and produced significantly smaller tumors when inoculated into SCID mice. In contrast, there was no significant reduction in the soft agar colony-forming ability or in vivo tumorigenicity of the DU-145 Krev-1 transfectants. These results suggest that the Krev-1 suppressor gene induces partial suppression of the malignant phenotype of human prostate cancer cells containing activated ras oncogenes.