A crucial event during angiogenesis is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. To investigate the possible role of proteases in endothelial cell invasiveness in vitro, bovine microvascular endothelial cells (BMEC) grown on collagen gels were treated with phorbol myristate acetate (PMA), a tumor promoter that markedly increases their production of collagenase and plasminogen activator. Whereas control BMEC were confined to the surface of the gels, PMA-treated BMEC invaded the underlying collagen matrix, where they formed an extensive network of capillary-like tubular structures. This phenomenon, which mimics some of the events occurring during angiogenesis in vivo, required protein synthesis and intercellular contact, was accompanied by collagen degradation, and was prevented by the metalloprotease inhibitor 1,10-phenanthroline.