We used Transwell chambers to study separately cellular motility and invasion. In order to assess the cellular motility, polycarbonate microporous filters were coated with extracellular matrix proteins which adsorbed on the filters without clogging the pores. To investigate the invasive behavior of tumor cells, filters were covered with a layer of Matrigel which clogged the pores. The motility and the invasion of breast cancer cell lines (MDA-MB-231, MCF-7/6 and MCF-7/AZ cells) were assessed quantitatively in different culture media: defined (serum-free), serum-containing and normal human fibroblast MRC-5 conditioned media. In serum-containing medium, tumor cells migrated and invaded through the coated and covered filters. Their motility and invasion potentials were considerably lower in defined medium, whereas medium conditioned by MRC-5 fibroblasts stimulated both motility and invasion but not growth. The MRC-5 conditioned medium induced also the spreading of clusters of MCF-7/6 cells grown on Matrigel-coated plates.