Two novel cDNA probes to chicken thrombospondin-1 (TSP-1) and TSP-3 were used to determine by in situ hybridization the origins of these extracellular matrix proteins during embryogenesis. Both TSP-1 and TSP-3 are expressed in embryonic cartilage. TSP-1 expression is limited to early chondrocytes, in contrast to TSP-3 mRNAs that are found in older proliferative and hypertrophic chondrocytes. TSP-1 and TSP-3 are expressed consecutively during neurogenesis as well, with the TSP-1 probe hybridizing in proliferating neuroblasts, and TSP-3 expressed by neurons as they are actively extending processes. A TSP-1 hybridization signal reappears in subpopulations of neurons in the spinal cord and brain after the periods of active neurite extension and programmed cell death are complete. TSP-1 is also expressed in the spinal cord floor plate, in mesenchyme surrounding the developing paramesonephric duct, at the tips of growing lung bronchioles, in lens, and in corneal endothelium. These observations indicate that there is little overlap in the expression patterns of TSP-1 and TSP-3 and that they are expressed consecutively during the development of cartilage and neurons. The pattern of TSP-1 expression in avascular tissues and in the floor plate is consistent with possible roles for this glycoprotein in regulating angiogenesis and establishing morphogenetic gradients.