F-spondin is a secreted protein expressed at high levels by the floor plate cells. The C-terminal half of the protein contains six thrombospondin type 1 repeats, while the N-terminal half exhibited virtually no similarity to any other protein until recently, when a Drosophila gene termed M-spondin was cloned; its product was found to share two conserved domains with the N-terminal half of F-spondin. We report the molecular cloning of four zebrafish genes encoding secreted proteins with these conserved domains. Two are zebrafish homologs of F-spondin, while the other two, termed mindin1 and mindin2, encode mutually related novel proteins, which are more related to the Drosophila M-spondin than to F-spondin. During embryonic development, all four genes are expressed in the floor plate cells. In addition to the floor plate, mindin1 is expressed in the hypochord cells, while mindin2 is expressed in the sclerotome cells. When ectopically expressed, Mindin proteins selectively accumulate in the basal lamina, suggesting that Mindins are extracellular matrix (ECM) proteins with high affinity to the basal lamina. We also report the spatial distribution of one of the F-spondin proteins, F-spondin2. F-spondin2 is localized to the thread-like structure in the central canal of the spinal cord, which is likely to correspond to Reissner's fiber known to be present in the vertebrate phylum. In summary, our study has defined a novel gene family of ECM molecules in the vertebrate, all of which may potentially be involved in development of the midline structure.