Fibulin-1 and fibulin-2 are two novel rod-like proteins which occur either in basement membranes or in interstitial fibrils in close association with fibronectin. They were examined for their sensitivity to proteolysis by matrix metalloproteinases (stromelysin, matrilysin), circulating proteases (thrombin, plasmin, kallikrein), leucocyte elastase and mast cell chymase. Fibulin-1 (95 kDa) was readily cleaved by leucocyte elastase, weakly by matrilysin and not by the other proteases. Cleavage occurred in a domain-connecting link region close to the N-terminus, giving rise to fragments of 70 kDa and 26 kDa. A much more extensive cleavage by all seven proteases was observed for fibulin-2 (195 kDa), giving rise to many fragments in the range 15-150 kDa. Vulnerable sites included two central link regions, the cysteine-free part of the large N-terminal globular domain but also several regions of epidermal-growth-factor(EGF)-like repeats which are a major part of the rod-like domain. The latter domain became much more sensitive to proteolysis in the presence of EDTA, demonstrating that calcium is required for stabilization. Edman degradation demonstrated cleavage of peptide bonds corresponding to the known specificities of these proteases. A similar proteolysis was also observed for fibulin-2 deposited by cultured fibroblasts into a dense fibrillar network. Since fibulin-2 is an abundant component of small and large blood vessels it could be a major target for proteolysis during vascular injuries.