During the crucial stages of tumor cell invasion and metastasis, neoplastic cells must traverse extracellular matrices for their migration to distant sites. Because basement membranes (BM) serve as a critical barrier to such passages, most previous in vitro assay models have utilized either an intact BM or a reconstituted rodent or avian BM-matrix to study this process. We have created a gel-like extracellular matrix derived from human amnions which contained type IV collagen, laminin, entactin, tenascin and heparan sulfate proteoglycan. This matrix, which we called Amgel, was used to study selected steps of invasion including cell attachment to matrix, degradation of it by proteolytic enzymes and movement of human tumor cells through matrix defects. An efficient tumor invasion assay system was developed utilizing filter-supported uniform coatings of this matrix in chambers. Human tumor cells (HT-1080 fibrosarcoma and RL-95 adenocarcinoma), when seeded onto Amgel-coated membranes, attached to matrix within 2 h and initiated a time-dependent migration and invasion process, as verified by biochemical analysis and both light and electron microscopy. In an optimized invasion assay 12-15% of tumor cells completely traversed the matrix during a 72-h period with > 90% viability. In contrast to these highly-invasive cells, normal human foreskin fibroblasts and normal human endometrial stromal cells exhibited minimal migration/matrix penetration during the same time period. When the Amgel-selected tumor cells (i.e. those penetrating the barrier) were isolated, subcultured, and re-exposed to Amgel, they had heightened invasiveness (2-3-fold) as compared to the parental cells. Thus, this improved 'all human' system for quantitating the invasive ability of tumor cells may provide a valuable tool in dissecting out the mechanistic underpinnings of human metastasis. In addition, this assay has the ability to screen agents which have potential anti-invasive and by extension anti-metastatic, activity or chemotactic properties.