The DNA sequence of the coding region of the mouse thrombospondin (TSP) 3 gene has been determined by analysis of both genomic and cDNA clones. Like TSP1 and TSP2, TSP3 has a homologous COOH-terminal domain and seven type III (Ca(2+)-binding) repeats. However, TSP3 contains four, rather than three, type II repeats and lacks the type I (TSP or properdin) repeats and procollagen homology characteristic of TSP1 and TSP2. In addition, the NH2-terminal domain of TSP3 differs markedly, both in sequence and in exon/intron structure, from that in TSP1 or TSP2. The gene is located on mouse chromosome 3, bands E3-F1, immediately upstream from the Muc1 (episialin) gene and is expressed in the developing mouse in a pattern that also differs from that of TSP1 or TSP2. Based on its structure, we suggest that TSP3 may play both a unique role in cell-matrix interactions and perform functions that overlap with those of TSP1 and TSP2.