The thrombospondins are a family of related glycoproteins found in the embryonic extracellular matrix. To date, five members of this family have been identified. Thrombospondin-1 and thrombospondin-2 have similar primary structure, but are expressed in different tissues at different times during development. Thrombospondins-3, -4, and cartilage oligomeric protein belong to a second thrombospondin subgroup in which the carboxyl-half of each molecule is most similar to thrombospondin-1 and -2. Here, we report the cloning and sequencing of a novel probe to avian thrombospondin-4. We have used this probe to determine the origins of thrombospondin-4 in the chick embryo by in situ hybridization. Thrombospondin-4 transcripts first appear in the mesenchyme surrounding bone anlage coinciding with the initial stages of osteogenesis. The expression in osteogenic tissues is transient: thrombospondin-4 mRNAs are not seen in the osteoblasts of bone collars in developing long bones. This pattern is distinct from avian thrombospondin-2, which is expressed in perichondrium and embryonic fibrous connective tissues. Our observations indicate that connective tissues are the principal site of thrombospondin-4 expression in the chick. The diverse origins of different thrombospondin gene family members imply distinctive roles for these proteins related to the growth and differentiation of cartilage, tendons, and bone. ©1995 Wiley-Liss, Inc.