Signal transduction from tyrosine kinase receptors mediates growth regulation of breast cancer cells in part through the GTPase Ras and downstream kinases. Rsu-1 is a cDNA previously identified as an inhibitor of Ras-induced transformation. An HA-epitope tagged Rsu-1 cDNA was introduced into the MCF7 breast carcinoma cell line. Stable transfectants were selected and used for analysis of Rsu-1 expression on growth control and Ras-dependent kinase pathways. Assessment of biological activity of HA-Rsu-1 transfectants revealed that HA-Rsu-1 clones showed slower anchorage dependent growth rates than control MCF7 cell lines and a significant reduction in anchorage independent growth. Analysis of cell cycle regulatory proteins required for transit through G1 revealed that HA-Rsu-1 transfectant cell lines expressed elevated levels of p21CIP CDK inhibitor. Perturbations in signal transduction pathways which can be activated by Ras were detected in the Ha-Rsu-1 transfectants. Exposure of serum-starved cells to EGF revealed that expression of HA-Rsu-1 increased ERK-2 kinase activation, decreased activation of Jun kinase and inhibited Rho-dependent Rho-alpha kinase (ROK) activity compared to control cells. While serum starvation reduced AKT activity to undetectable levels in HA-Rsu-1 transfectants but not in control MCF7 cells, activation of AKT kinase by serum was unaffected by HA-Rsu-1 expression. Finally, the level of c-myc transcription in HA-Rsu-1 transfectants reached only 60% of the MCF7 control cell line following serum stimulation of starved cells while Fos RNA levels were similar to control cells. These results demonstrate that increased Rsu-1 expression critically altered cell cycle regulation and growth of MCF7 cells as well as signaling pathways in MCF7 cells required for malignant growth.