We have cloned the cDNAs of the zebrafish (Danio rerio) members of the matrilin family of extracellular adaptor proteins. In contrast to mammals, no orthologue of matrilin-2 was found in zebrafish, either by RT (reverse-transcriptase) PCR using degenerated primers or by screening the databases (Ensembl and NCBI); however, two forms of matrilin-3, matrilin-3a and -3b, were present. The identity with the mammalian matrilins is from more than 70% for the VWA (von Willebrand factor A)-like domains to only 28% for the coiled-coil domains of matrilin-3a and -3b. In all zebrafish matrilins we found a greater variety of splice variants than in mammals, with splicing mainly affecting the number of EGF (epidermal growth factor)-like repeats. The exon-intron organization is nearly identical with that of mammals, and also the characteristic AT-AC intron interrupting the exons coding for the coiled-coil domain is conserved. In the matrilin-3b gene a unique exon codes for a proline- and serine/threonine-rich domain, possibly having mucin-like properties. The matrilin-1 and -3a genes were mapped to chromosome 19 and 20 respectively by the radiation hybrid method. The temporal and spatial expression of zebrafish matrilins is similar to that seen in the mouse. Zebrafish matrilin-4 is highly expressed as early as 24 hpf (h post fertilization), whereas the other matrilins show peak expression at 72 hpf. By immunostaining of whole mounts and sections, we found that matrilin-1 and -3a show predominantly skeletal staining, whereas matrilin-4 is more widespread, with the protein also being present in loose connective tissues and epithelia.