Fibulin-1 and fibulin-2 have previously been identified as basement membrane and microfibrillar proteins with a broad binding repertoire for other extracellular ligands. Here we report on the cloning and sequence analysis of human fibulin-3 (487 residues), also known as protein S1-5, and fibulin-4 (443 residues). These novel members of this protein family are most closely related to fibulin-1C. They consist of a C-terminal globular domain III, also shared by the fibrillins, a central rod-like element composed of five calcium-binding epidermal growth factor-like (EG) modules (domain II) and an N-terminal interrupted EG module (domain I) which replaces the anaphylatoxin-like modules of the other fibulins. This predicted domain structure was supported by electron microscopy of fibulin-4, which demonstrated short rods. Northern blots showed that both novel fibulins are expressed in several human tissues to a variable extent and that they are up-regulated in quiescent fibroblasts. Specific antibodies which were raised against each of the novel fibulins did not cross-react with fibulin-1. Immunohistology of adult mouse tissues showed that fibulin-3, fibulin-4 and fibulin-1 have overlapping but distinct extracellular tissue localizations. A particularly prominent feature was the staining of variable sets of large and small blood vessels.