Thrombospondin 1 is a secreted, trimeric glycoprotein that mediates interactions between cells and extracellular matrix and exhibits cell-specific effects on migration and proliferation. Recently, two additional thrombospondin genes (thrombospondin 2 and 3) have been identified. To study the functions of these proteins, we have used in situ hybridization and RNAse protection assays to compare the expression of the genes encoding thrombospondin 1, 2, and 3 during murine embryogenesis. Thrombospondin mRNAs were associated with ossification, neuronal organogenesis, and lung development, although transcripts were differentially expressed. Thrombospondin 1 was predominant from days 10 to 13. During this period, high but transient levels of expression were observed in the neural tube, head mesenchyme, and cardiac cushions. In contrast, a more constant level of thrombospondin 1 mRNA was apparent in resident megakaryocytes of the liver, as well as in circulating megakaryocytes; neither thrombospondin 2 nor 3 was detected in these cells. Thrombospondin 1 was also produced by cells of the developing kidney and gut. The expression of thrombospondin 2 was confined principally to organized connective tissue that included pericardium, pleura, perichondrium, periosteum, meninges, ligaments, and reticular dermis. Thrombospondin 2 was also produced by differentiating skeletal myoblasts and by cells of the kidney and gut. Moreover, high levels of expression were detected in blood vessels. Thrombospondin 3 mRNA was restricted to brain, cartilage, and lung. Although thrombospondin 1, 2, and 3 belong to a family of structurally related genes, the differences observed in the spatiotemporal distribution of the corresponding mRNAs indicate unique functions for these secreted proteins.