CAS proteins and Ezrin, Radixin, Moesin (ERM) family members act as intracellular scaffolds and are involved in interactions with the cytoskeleton, respectively. Both protein families have previously been associated with metastasis and poor prognosis in cancer. Our group recently reported on the overexpression of EZR/VIL2 and BCAR1 and their protein products in breast carcinoma effusions compared to primary breast carcinoma. In the present study, the role of these two proteins was studied in semi-normal MCF10A cells and metastatic MDA-MB-231 breast carcinoma cells cultured in tri-dimensional (3-D) conditions that were hypothesized to reproduce the in vivo conditions of breast cancer metastasis. MCF10A cells formed spheroid-shaped colonies without any Matrigel invasion, while MDA-MB-231 cells displayed an invasive phenotype and showed satellite projections that bridged multiple cell colonies in 3-D culture. E-cadherin was expressed in MCF10A, but not in MDA-MB-231 cells. The temporal expression of ezrin and BCAR1/p130Cas at the mRNA and protein level differed in the two cell lines upon 3-D culturing on Matrigel. Upregulation of BCAR1/p130cas was observed in the transition of MDA-MB-231 from attached to detached culture. Silencing of Ezrin and p130Cas in MDA-MB-231 cells by short hairpin RNA resulted in decreased invasive potential, and p130Cas silencing further resulted in smaller spheroid/colony formation. Our data show that MCF10A and MDA-MB-231 cells differ in their ability to form spheroids, in expression of E-cadherin and in the expression of Ezrin and BCAR1/p130Cas in 3-D cultures on Matrigel, suggesting a role in tumor progression in breast carcinoma.